Poly(amino acid) targeting moieties

ABSTRACT

The present invention generally relates to polymers and macromolecules, in particular, to polymers useful in particles such as nanoparticles. One aspect of the invention is directed to a method of developing nanoparticles with desired properties. In one set of embodiments, the method includes producing libraries of nanoparticles having highly controlled properties, which can be formed by mixing together two or more macromolecules in different ratios. One or more of the macromolecules may be a polymeric conjugate of a moiety to a biocompatible polymer. In some cases, the nanoparticle may contain a drug. Other aspects of the invention are directed to methods using nanoparticle libraries.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/910,097, Attorney Docket No. BBZ-011-1, filed Apr. 4, 2007, titled“Amphiphilic compound assisted polymeric particles for targeteddelivery;” U.S. Provisional Application No. 60/985,104, Attorney DocketNo. BBZ-011-2, filed Nov. 2, 2007, titled “Lipid-Stabilized PolymericNanoparticles for Targeted Drug Delivery;” U.S. Provisional ApplicationNo. 60/938,590, Attorney Docket No. BBZ-012-1, filed May 17, 2007,titled “Poly(Amino Acid)-Targeted Drug Delivery;” U.S. ProvisionalApplication No. 60/986,202, Attorney Docket No. BBZ-012-2, filed Nov. 7,2007, titled “Poly(Amino Acid)-Targeted Drug Delivery;” and U.S.Provisional Application No. 60/990,250, Attorney Docket No. BBZ-012-3,filed Nov. 26, 2007, titled “Poly(Amino Acid)-Targeted Drug Delivery;”all of which are incorporated herein by reference in their entirety.Additionally, the contents of any patents, patent applications, andreferences cited throughout this specification are hereby incorporatedby reference in their entireties.

FIELD OF INVENTION

The present invention generally relates to targeted nanoparticles thattarget tissue basement membrane.

BACKGROUND

The delivery of a drug to a patient with controlled-release of theactive ingredient has been an active area of research for decades andhas been fueled by the many recent developments in polymer science. Inaddition, controlled release polymer systems can be designed to providea drug level in the optimum range over a longer period of time thanother drug delivery methods, thus increasing the efficacy of the drugand minimizing problems with patient compliance.

Nanoparticles have been developed as vehicles used in the administrationfor the delivery of small molecule drugs as well as proteins, peptidedrugs and nucleic acids. The drugs are typically encapsulated orconjugated in a polymer matrix which is biodegradable and biocompatible.As the polymer is degraded and/or as the drug diffuses out of thepolymer, the drug is released into the body. Typically, polymers used inpreparing these particles are polyesters such aspoly(lactide-co-glycolide) (PLGA), polyglycolic acid,poly-beta-hydroxybutyrate, polyacrylic acid ester, polymetacrylate,polyglutamate, etc. These particles can also protect the drug fromdegradation by the body. Furthermore, these particles can beadministered using a wide variety of administration routes.

Targeting controlled release polymer systems (e.g., targeted to aparticular tissue or cell type or targeted to a specific diseased tissuebut not normal tissue) is desirable because it reduces the amount of adrug present in tissues of the body that are not targeted. This isparticularly important when treating a condition such as cancer where itis desirable that a cytotoxic dose of the drug is delivered to cancercells without killing the surrounding non-cancerous tissue. Effectivedrug targeting should reduce the undesirable and sometimes lifethreatening side effects common in anticancer therapy.

Targeted delivery for diagnosis and therapeutic applications has untilrecently largely been limited to receptor ligands such as antibodies,modified-antibodies and nucleic acids. Antibodies are the most widelyused type of targeting agent today. The large size of antibody moleculescan be advantageous for bimodal binding mechanisms but it may also leadto poor solid penetration and slow elimination from the bloodcirculation. Unfortunately, slow elimination kinetics can causemyelotoxicity. In addition, its in vivo application has been proven morechallenging because of cost and potential immunogenicity after repeatinjections of such formulations. To avoid these problems, Fab's and scFvhave successfully been made but are still too large. The molecularweight of fragments has been shown to be a major factor of capillarypermeability, so fragments can reach the interstitial spaces more easilythan whole antibody. However, the effects of the increased permeabilityare offset by the more rapid excretion of the antibody fragments whichdecreases the ability of the antibody to cross membranes resulting inlower absolute tumor levels as well as lower blood and tissue levels.

Accordingly, there is a need for developing new and alternativetargeting controlled release polymer systems, especially those useful inthe treatment of diseases, e.g., cancer, restenosis, and vulnerableplaques.

SUMMARY OF THE INVENTION

There remains a need for compositions useful in the treatment orprevention or amelioration of one or more symptoms of cancer andcardiovascular disease. There also remains a need for compositionsuseful in the treatment or prevention or amelioration of one or moresymptoms of vulnerable plaques. In one aspect, the invention provides acontrolled-release system, comprising a plurality of target-specificstealth nanoparticles; wherein said nanoparticles contain targetingmoieties attached thereto, wherein the targeting moiety targets thetissue basement membrane, such as the vascular basement membrane.

In one aspect, the invention provides a controlled-release system,comprising a plurality of target-specific stealth nanoparticles; whereinsaid nanoparticles contain targeting moieties attached thereto, whereinthe targeting moiety is a poly(amino acid) that targets the tissuebasement membrane, such as the vascular basement membrane. In oneembodiment, the nanoparticle has an amount of targeting moiety effectivefor the treatment of vulnerable plaque in a subject in need thereof. Inanother embodiment, the nanoparticle has an amount of targeting moietyeffective for the treatment of cancer in a subject in need thereof. Asdiscussed below, the nanoparticle has an amount of targeting moietyeffective for the treatment of restinosis in a subject in need thereof.In one embodiment, the cancer to be treated is selected from the groupconsisting of breast cancer, ovarian cancer, brain cancer, colon cancer,renal cancer, lung cancer, bladder cancer, prostate cancer and melanoma.In still another embodiment, the cancer is breast cancer.

In one embodiment, the nanoparticles of the invention can be used totreat ovarian cancer, breast cancer, and human pancreatic cancer in asubject in need thereof.

In one embodiment of the controlled-release system of the invention, thepoly(amino acid) comprises natural amino acids, unnatural amino acids,modified amino acids, protected amino acids or mimetic of amino acids.In still another embodiment, the poly(amino acid) is selected from thegroup consisting of a glycoprotein, protein, peptidomimetic, affibody orpeptide. In yet another embodiment, the poly(amino acid) binds to thebasement membrane of tissues, such as the vascular basement of a bloodvessel. In another embodiment, the poly(amino acid) binds to collagen.In still another embodiment, the poly(amino acid) binds to collagen IV.

In one embodiment, the poly(amino acid) is an affibody, wherein theaffibody is an anti-HER2 affibody. In another embodiment, the poly(aminoacid) is a peptide, wherein the peptide comprises a sequence selectedfrom the group consisting of AKERC, CREKA, ARYLQKLN and AXYLZZLN,wherein X and Z are variable amino acids.

In one embodiment of the controlled-release system of the invention, thenanoparticle comprises a polymeric matrix. In one embodiment, thepolymeric matrix comprises two or more synthetic or natural polymers. Ina particular embodiment, the polymeric matrix comprises polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, or polyamines,polyglutamate, dextran, or combinations thereof.

In another embodiment, the polymeric matrix comprises one or morepolyesters, polyanhydrides, polyethers, polyurethanes,polymethacrylates, polyacrylates or polycyanoacrylates. In anotherembodiment, at least one polymer is a polyalkylene glycol. In stillanother embodiment, the polyalkylene glycol is polyethylene glycol. Inyet another embodiment, at least one polymer is a polyester. In oneembodiment, the polyester is selected from the group consisting ofpoly-lactic-co-glycolic acid (PLGA), polylactic acid (PLA), polyglycolicacid (PGA), and polycaprolactones. In still another embodiment, thepolyester is PLGA or PLA.

In one embodiment of the controlled-release system of the invention, thenanoparticle comprises a polymeric matrix, wherein the polymeric matrixcomprises a copolymer of two or more polymers. In another embodiment,the copolymer is a copolymer of a polyalkylene glycol and a polyester.In still another embodiment, the copolymer is a copolymer of PLGA andPEG. In yet another embodiment, the polymeric matrix comprises PLGA anda copolymer of PLGA and PEG.

In another embodiment of the controlled-release system of the invention,the nanoparticle comprises a polymeric matrix, wherein the polymericmatrix comprises a lipid-terminated polyalkylene glycol and a polyester.In one embodiment, the polymeric matrix comprises lipid-terminated PEGand PLGA. In one embodiment, the lipid is of the Formula V, and saltsthereof. In one one embodiment, the lipid is 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof.

In one embodiment of the controlled-release system, a portion of thepolymer matrix is covalently bound to the poly(amino acid). In anotherembodiment, the polymer matrix is covalently bound to the poly(aminoacid) via the free terminus of PEG. In still another embodiment, thepolymer matrix is covalently bound to the poly(amino acid) via acarboxyl group at the free terminus of PEG. In yet another embodiment,the polymer matrix is covalently bound to the poly(amino acid) via amaleimide functional group at the free terminus of PEG.

In one embodiment of the controlled-release system, the nanoparticle hasa ratio of ligand-bound polymer to non-functionalized polymer effectivefor the treatment of cancer. In another embodiment, the nanoparticle hasa ratio of ligand-bound polymer to non-functionalized polymer effectivefor the treatment of a vulnerable plaque. In still another embodiment,the polymers of the polymer matrix have a molecular weight effective forthe treatment of cancer. In another embodiment, the polymers of thepolymer matrix have a molecular weight effective for the treatment ofvulnerable plaque.

In another embodiment of the controlled-release system, the nanoparticlehas a surface charge effective for the treatment of cancer. In stillanother embodiment, the nanoparticle has a surface charge effective forthe treatment of vulnerable plaque. In yet another embodiment, saidsystem is suitable for target-specific treatment of a disease ordisorder and delivery of a therapeutic agent.

In another embodiment of the controlled-release system, the nanoparticlefurther comprises a therapeutic agent. In one embodiment, thetherapeutic agent is associated with the surface of, encapsulatedwithin, surrounded by, or dispersed throughout the nanoparticle. Inanother embodiment, the therapeutic agent is encapsulated within thehydrophobic core of the nanoparticle. In still another embodiment, thetherapeutic agent is selected from the group consisting of mitoxantrone,platin and docetaxel. In yet another embodiment, the therapeutic agentis selected from the group consisting of VEGF, fibroblast growthfactors, monocyte chemoatractant protein 1 (MCP-1), transforming growthfactor alpha (TGF-alpha), transforming growth factor beta (TGF-beta),DEL-1, insulin like growth factors (IGF), placental growth factor(PLGF), hepatocyte growth factor (HGF), prostaglandin E1 (PG-E1),prostaglandin E2 (PG-E2), tumor necrosis factor alpha (THF-alpha),granulocyte stimulating growth factor (G-CSF), granulocyte macrophagecolony-stimulating growth factor (GM-CSF), angiogenin, follistatin, andproliferin, PR39, PR11, nicotine, hydroxy-methylglutaryl coenzyme A (HMGCoA) reductase inhibitors, statins, niacin, bile acid resins, fibrates,antioxidants, extracellular matrix synthesis promoters, inhibitors ofplaque inflammation and extracellular degradation, and estradiol.

In another aspect, the invention provides a method of treating breastcancer in a subject in need thereof, comprising administering to thesubject an effective amount of the controlled-release system of theinvention. In one embodiment for the treatment of breast cancer, thecontrolled-release system is administered systemically. In still anotherembodiment, the controlled-release system is administered directly tobreast cancer cells. In another embodiment, the controlled-releasesystem is administered directly to breast cancer cells by injection intotissue comprising the breast cancer cells. In another embodiment, thecontrolled-release system is administered to the subject by implantationof nanoparticles at or near breast cancer cells during surgical removalof a tumor. In still another embodiment, the controlled-release systemis administered via intravenous administration.

In another aspect, the invention provides a method of treatingvulnerable plaque in a subject in need thereof, comprising administeringto the subject an effective amount of the controlled-release system ofthe invention. In one embodiment, the controlled-release system islocally administered to a designated region of the blood vessel wherethe vulnerable plaque occurs. In still another embodiment, thecontrolled-release system is administered via a medical device. In yetanother embodiment, the medical device is a drug eluding stent, needlecatheter, or stent graft.

In another aspect, the invention provides a method of treatingrestenosis in a subject in need thereof, comprising administering to thesubject an effective amount of the controlled-release system of theinvention. In one embodiment, the controlled-release system is locallyadministered to a designated region of the blood vessel where therestenosis occurs. In still another embodiment, the controlled-releasesystem is administered via a medical device. In yet another embodiment,the medical device is a drug eluding stent, needle catheter, or stentgraft. In another embodiment, the invention provides a method oftreating restenosis in a subject in need thereof, comprisingadministering to the subject an effective amount of thecontrolled-release system of the invention wherein the controlledrelease system contains a drug suitable for the treatment of restenosis.In another embodiment, the invention provides a method of treatingrestenosis in a subject in need thereof, comprising administering to thesubject an effective amount of the controlled-release system of theinvention wherein the controlled release system contains at least twodrugs suitable for the treatment of restenosis. In another embodiment,the invention provides a method of treating restenosis in a subject inneed thereof, comprising administering to the subject an effectiveamount of the controlled-release system of the invention wherein thecontrolled release system contains zotarolimus and dexamethasone.

In one embodiment, the nanoparticles of this invention are deliveredlocally to the coronary arteries, central arteries, peripheral arteries,veins, and bile ducts. In another embodiment, the nanoparticles of thisinvention are delivered locally to the coronary arteries, centralarteries, peripheral arteries, veins, and bile ducts after theimplantation of a stent in such tissue in a patient for the treatment ofrestenosis. In another embodiment, the nanoparticles of this inventionare administered to a patient undergoing a coronary angioplasty, aperipheral angioplasty, a renal artery angioplasty, or a carotidangioplasty in order to prevent resenosis. In another embodiment, thenanoparticles of this invention are administered within 12 hours of apatient undergoing a coronary angioplasty, a peripheral angioplasty, arenal artery angioplasty, or a carotid angioplasty in order to preventresenosis. In another embodiment, the nanoparticles of this inventionare administered locally to a patient undergoing a coronary angioplasty,a peripheral angioplasty, a renal artery angioplasty, or a carotidangioplasty in order to prevent resenosis.

In one embodiment, the nanoparticles of this invention pass through theendothelial layer of a blood vessel due to plaque damage of theendothelial tissue and bind to collage 4 of the basement membrane.

In another aspect, the invention provides a method of preparing astealth nanoparticle, wherein the nanoparticle has a ratio ofligand-bound polymer to non-functionalized polymer effective for thetreatment of a disease, comprising: providing a therapeutic agent;providing a polymer; providing a poly(amino acid) ligand; mixing thepolymer with the therapeutic agent to prepare particles; and associatingthe particles with the poly(amino acid) ligand. In one embodiment of themethod, the polymer comprises a copolymer of two or more polymers. Inanother embodiment, the copolymer is a copolymer of PLGA and PEG.

In another aspect, the invention provides a method of preparing astealth nanoparticle, wherein the nanoparticle has a ratio ofligand-bound polymer to non-functionalized polymer effective for thetreatment of a disease, comprising: providing a therapeutic agent;providing a first polymer; providing a poly(amino acid) ligand; reactingthe first polymer with the poly(amino acid) ligand to prepare aligand-bound polymer; and mixing the ligand-bound polymer with a second,non-functionalized polymer, and the therapeutic agent; such that thestealth nanoparticle is formed. In one embodiment of this method, thefirst polymer comprises a copolymer of two or more polymers. In anotherembodiment, the second, non-functionalized polymer comprises a copolymerof two or more polymers. In another embodiment of this method, the firstpolymer is first reacted with a lipid, to form a polymer/lipidconjugate, which is then reacted with the poly(amino acid). In stillanother embodiment, the lipid is 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof.In yet another embodiment, the copolymer is a copolymer of PLGA and PEG.In still another embodiment, the first polymer is a copolymer of PLGAand PEG, wherein the PEG has a carboxyl group at the free terminus.

In one embodiment of the aforementioned methods, the disease to betreated is cancer, vulnerable plaque, or restenosis.

In another aspect, the invention provides a stealth nanoparticle,comprising a copolymer of PLGA and PEG; and a therapeutic agentcomprising mitoxantrone, platin or docetaxel; wherein said nanoparticlecontains targeting moieties attached thereto, wherein the targetingmoiety is an anti-HER2 affibody.

In yet another aspect, the invention provides a stealth nanoparticle,comprising a copolymer of PLGA and PEG; and a therapeutic agent; whereinsaid nanoparticle contains targeting moieties attached thereto, whereinthe targeting moiety comprises AKERC or CREKA.

In another aspect, the invention provides a stealth nanoparticle,comprising a polymeric matrix comprising a complex of a phospholipidbound-PEG and PLGA; and a therapeutic agent; wherein said nanoparticlecontains targeting moieties attached thereto, wherein the targetingmoiety is a poly(amino acid).

In still another aspect, the invention provides a controlled-releasesystem, comprising a plurality of target-specific stealth nanoparticles;wherein said nanoparticles contain targeting moieties attached thereto,wherein the targeting moiety is a basement membrane-targeting moiety. Inone embodiment, the basement membrane-targeting moiety is AKERC, CREKA,ARYLQKLN or AXYLZZLN, wherein X and Z are variable amino acids.

In another embodiment of the controlled-release system of the invention,the polymeric matrix is surrounded by a lipid monolayer shell. In oneembodiment, the lipid monolayer shell comprises an amphiphilic compound.In another embodiment, the amphiphilic compound is lecithin. In anotherembodiment, the lipid monolayer is stabilized.

In another aspect, the invention provides a controlled-release system,comprising a plurality of target-specific stealth nanoparticles; whereinsaid nanoparticles contain targeting moieties attached thereto, whereinthe targeting moiety is a poly(amino acid). In one embodiment, thenanoparticles target antigen presenting cells and elicit animmunomodulatory response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 2 show representative synthesis schematics for thetarget-specific stealth nanoparticle of the invention.

FIG. 3 is a representative schematic of a nanoparticle of the invention.

FIG. 4 shows a schematic illustration of amphiphilic compound assistedpolymeric nanoparticles for targeted drug delivery.

FIGS. 5A and 5B show size and zeta-potential stabilities, respectively,for nanoparticles prepared according to a process of the invention.

FIGS. 6 and 7 demonstrate drug encapsulation efficiency of a lipidassisted polymeric nanoparticle as compared with a non-lipid assistedpolymeric nanoparticle.

FIG. 8 shows a drug release profile for a nanoparticle preparedaccording to a process of the invention.

FIGS. 9A and 9B demonstrate a lecithin concentration effect onPLGA-Lipid-PEG nanoparticle size and zeta potential, respectively.

FIG. 10 demonstrates a schematic illustration of a CREKA-targetedPLGA-Lipid-PEG nanoparticle.

FIGS. 11A and 11B demonstrate that (A) CREKA-targeted PLGA-Lipid-PEGnanoparticles effectively bind to collagen IV coated surface and (B)bare (nontargeted) PLGA-Lipid-PEG nanoparticles rarely bind to collagenIV coated surface.

FIGS. 12A and 12B demonstrate (A) H&E staining of normal rat aorta; (B)H&E staining of balloon injured aorta (endothelium layer was removed).

FIGS. 13A and 13B demonstrates that CREKA-targeted PLGA-Lipid-PEGnanoparticles effectively bind to a balloon-injured rat aorta.

FIGS. 14A and 14B demonstrate that D-CREKA-targeted PLGA-Lipid-PEGnanoparticles (D-form of amino acids) do not bind to balloon-injured rataorta.

FIGS. 15A and 15B demonstrates that scrambled peptide CEAKR-targetedPLGA-Lipid-PEG nanoparticles do not bind to balloon-injured rat aorta.

FIGS. 16A and 16B demonstrate that CREKA-targeted PLGA-Lipid-PEGnanoparticles do not bind to a normal rat aorta.

FIG. 17 is a schematic illustration of CREKA-targeted PLGA-Lipid-PEGnanoparticle

FIG. 18 shows fluorescence images of ARYLQKLN-targeted PLGA-Lipid-PEGnanoparticles incubating with basement membrane proteins for 10 minutes:(A) PBS; (B) Collagen I; (C) Collagen II; (D) Collagen IV; (E)Fibronectin; and (F) vitronectin.

FIG. 19 demonstrates that ARYLQKLN-targeted PLGA-Lipid-PEG nanoparticlesbind to a balloon-injured rat aorta.

FIG. 20 demonstrates that ARYLQKLN-targeted PLGA-Lipid-PEG nanoparticlesdo not bind to a normal rat aorta.

FIGS. 21A, 21B and 21C demonstrate the size diameter (<100 nm) anddistribution as visualized by electron microscopy of a nanoparticle ofthe invention; direct visualization of an affibody on the surface of ananoparticle of the invention carried out using fluorescent imaging; and¹H-NMR (proton nuclear magnetic resonance) spectrum of aPLA-PEG-affibody nanoparticle of the invention.

FIG. 22 shows fluorescent microscopy of nanoparticle-affibodybioconjugates incubated with HER-2 positive cell lines.

FIG. 23 shows combined fluorescent images (60× magnification) of asingle SK-BR-3 cell to reconstruct a three-dimensional image of a cell,demonstrating the internalization of targeted NP-affibody bioconjugatesto the cell.

FIG. 24 shows the results of a cell viability assay (MTS assay) toevaluate the differential toxicity of targeted (Np-Affb) and untargetednanoparticles (Np) with and without encapsulated paclitaxel (Ptxl).

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to particles, and, inparticular, nanoparticles, wherein the nanoparticles comprise a drugdelivery system for the targeted delivery of a therapeutic agent.

In one embodiment, the nanoparticle of the controlled release system hasan amount of targeting moiety (i.e., a poly(amino acid)) effective forthe targeting of tissue basement membrane. In one embodiment, thenanoparticle of the controlled release system has an amount of targetingmoiety (i.e., a poly(amino acid)) effective for the targeting ofvascular basement membrane. In certain embodiments, the poly(amino acid)is conjugated to a polymer, and the nanoparticle comprises a certainratio of ligand-conjugated polymer to non-functionalized polymer. Thenanoparticle can have an optimized ratio of these two polymers, suchthat an effective amount of ligand is associated with the nanoparticlefor treatment of a disease, e.g., cancer (e.g., breast cancer),vulnerable plaque, restenosis. For example, increased ligand density(e.g., on a PLGA-PEG copolymer) will increase target binding (cellbinding/target uptake), making the nanoparticle “target specific.”Alternatively, a certain concentration of non-functionalized polymer(e.g., non-functionalized PLGA-PEG copolymer) in the nanoparticle cancontrol inflammation and/or immunogenicity (i.e., the ability to provokean immune response), and allow the nanoparticle to have a circulationhalf-life that is therapeutically effective for the treatment of, e.g.,cancer, vulnerable plaque, or restenosis. Furthermore, thenon-functionalized polymer can lower the rate of clearance from thecirculatory system via the reticuloendothelial system. Thus, thenon-functionalized polymer gives the nanoparticle “stealth”characteristics. Additionally, the non-functionalized polymer balancesan otherwise high concentration of ligands, which can otherwiseaccelerate clearance by the subject, resulting in less delivery to thetarget cells.

Target-Specific Stealth Nanoparticles Comprising Polymers

In preferred embodiments, the nanoparticles of the invention comprise amatrix of polymers. In general, a “nanoparticle” refers to any particlehaving a diameter of less than 1000 nm. In some embodiments, atherapeutic agent and/or targeting moiety (i.e., a poly(amino acid)) canbe associated with the polymeric matrix. In some embodiments, thetargeting moiety can be covalently associated with the surface of apolymeric matrix. In some embodiments, covalent association is mediatedby a linker. In some embodiments, the therapeutic agent can beassociated with the surface of, encapsulated within, surrounded by,and/or dispersed throughout the polymeric matrix.

A wide variety of polymers and methods for forming particles therefromare known in the art of drug delivery. In some embodiments of theinvention, the matrix of a particle comprises one or more polymers. Anypolymer may be used in accordance with the present invention. Polymersmay be natural or unnatural (synthetic) polymers. Polymers may behomopolymers or copolymers comprising two or more monomers. In terms ofsequence, copolymers may be random, block, or comprise a combination ofrandom and block sequences. Typically, polymers in accordance with thepresent invention are organic polymers.

A “polymer,” as used herein, is given its ordinary meaning as used inthe art, i.e., a molecular structure comprising one or more repeat units(monomers), connected by covalent bonds. The repeat units may all beidentical, or in some cases, there may be more than one type of repeatunit present within the polymer. In some cases, the polymer isbiologically derived, i.e., a biopolymer. Non-limiting examples includepeptides or proteins (i.e., polymers of various amino acids), or nucleicacids such as DNA or RNA. In some cases, additional moieties may also bepresent in the polymer, for example biological moieties such as thosedescribed below.

If more than one type of repeat unit is present within the polymer, thenthe polymer is said to be a “copolymer.” It is to be understood that inany embodiment employing a polymer, the polymer being employed may be acopolymer in some cases. The repeat units forming the copolymer may bearranged in any fashion. For example, the repeat units may be arrangedin a random order, in an alternating order, or as a “block” copolymer,i.e., comprising one or more regions each comprising a first repeat unit(e.g., a first block), and one or more regions each comprising a secondrepeat unit (e.g., a second block), etc. Block copolymers may have two(a diblock copolymer), three (a triblock copolymer), or more numbers ofdistinct blocks.

It should be understood that, although the terms “first,” “second,” etc.may be used herein to describe various elements, including polymericcomponents, these terms should not be construed as being limiting (e.g.,describing a particular order or number of elements), but rather, asbeing merely descriptive, i.e., labels that distinguish one element fromanother, as is commonly used within the field of patent law. Thus, forexample, although one embodiment of the invention may be described ashaving a “first” element present and a “second” element present, otherembodiments of the invention may have a “first” element present but no“second” element present, a “second” element present but no “first”element present, two (or more) “first” elements present, and/or two (ormore) “second” elements present, etc., and/or additional elements suchas a “first” element, a “second” element, and a “third” element, withoutdeparting from the scope of the present invention.

Various embodiments of the present invention are directed to copolymers,which, in particular embodiments, describes two or more polymers (suchas those described herein) that have been associated with each other,usually by covalent bonding of the two or more polymers together. Thus,a copolymer may comprise a first polymer and a second polymer, whichhave been conjugated together to form a block copolymer where the firstpolymer is a first block of the block copolymer and the second polymeris a second block of the block copolymer. Of course, those of ordinaryskill in the art will understand that a block copolymer may, in somecases, contain multiple blocks of polymer, and that a “block copolymer,”as used herein, is not limited to only block copolymers having only asingle first block and a single second block. For instance, a blockcopolymer may comprise a first block comprising a first polymer, asecond block comprising a second polymer, and a third block comprising athird polymer or the first polymer, etc. In some cases, block copolymerscan contain any number of first blocks of a first polymer and secondblocks of a second polymer (and in certain cases, third blocks, fourthblocks, etc.). In addition, it should be noted that block copolymers canalso be formed, in some instances, from other block copolymers.

For example, a first block copolymer may be conjugated to anotherpolymer (which may be a homopolymer, a biopolymer, another blockcopolymer, etc.), to form a new block copolymer containing multipletypes of blocks, and/or to other moieties (e.g., to non-polymericmoieties).

In some embodiments, the polymer (e.g., copolymer, e.g., blockcopolymer) is amphiphilic, i.e., having a hydrophilic portion and ahydrophobic portion, or a relatively hydrophilic portion and arelatively hydrophobic portion. A hydrophilic polymer is one generallythat attracts water and a hydrophobic polymer is one that generallyrepels water. A hydrophilic or a hydrophobic polymer can be identified,for example, by preparing a sample of the polymer and measuring itscontact angle with water (typically, the polymer will have a contactangle of less than 60°, while a hydrophobic polymer will have a contactangle of greater than about 60°). In some cases, the hydrophilicity oftwo or more polymers may be measured relative to each other, i.e., afirst polymer may be more hydrophilic than a second polymer. Forinstance, the first polymer may have a smaller contact angle than thesecond polymer.

In one set of embodiments, a polymer (e.g., copolymer, e.g., blockcopolymer) of the present invention includes a biocompatible polymer,i.e., the polymer that does not typically induce an adverse responsewhen inserted or injected into a living subject, for example, withoutsignificant inflammation and/or acute rejection of the polymer by theimmune system, for instance, via a T-cell response. Accordingly, thenanoparticles of the present invention can be “non-immunogenic.” Theterm “non-immunogenic” as used herein refers to endogenous growth factorin its native state which normally elicits no, or only minimal levelsof, circulating antibodies, T-cells, or reactive immune cells, and whichnormally does not elicit in the individual an immune response againstitself.

It will be recognized, of course, that “biocompatibility” is a relativeterm, and some degree of immune response is to be expected even forpolymers that are highly compatible with living tissue. However, as usedherein, “biocompatibility” refers to the acute rejection of material byat least a portion of the immune system, i.e., a non-biocompatiblematerial implanted into a subject provokes an immune response in thesubject that is severe enough such that the rejection of the material bythe immune system cannot be adequately controlled, and often is of adegree such that the material must be removed from the subject. Onesimple test to determine biocompatibility is to expose a polymer tocells in vitro; biocompatible polymers are polymers that typically willnot result in significant cell death at moderate concentrations, e.g.,at concentrations of 50 micrograms/10⁶ cells. For instance, abiocompatible polymer may cause less than about 20% cell death whenexposed to cells such as fibroblasts or epithelial cells, even ifphagocytosed or otherwise uptaken by such cells. Non-limiting examplesof biocompatible polymers that may be useful in various embodiments ofthe present invention include polydioxanone (PDO), polyhydroxyalkanoate,polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide,polylactide, PLGA, polycaprolactone, or copolymers or derivativesincluding these and/or other polymers.

In certain embodiments, the biocompatible polymer is biodegradable,i.e., the polymer is able to degrade, chemically and/or biologically,within a physiological environment, such as within the body. As usedherein, “biodegradable” polymers are those that, when introduced intocells, are broken down by the cellular machinery (biologicallydegradable) and/or by a chemical process, such as hydrolysis,(chemically degradable) into components that the cells can either reuseor dispose of without significant toxic effect on the cells.

In a preferred embodiment, the biodegradable polymer and theirdegradation byproducts are biocompatible.

For instance, the polymer may be one that hydrolyzes spontaneously uponexposure to water (e.g., within a subject), the polymer may degrade uponexposure to heat (e.g., at temperatures of about 37° C.). Degradation ofa polymer may occur at varying rates, depending on the polymer orcopolymer used. For example, the half-life of the polymer (the time atwhich 50% of the polymer is degraded into monomers and/or othernonpolymeric moieties) may be on the order of days, weeks, months, oryears, depending on the polymer. The polymers may be biologicallydegraded, e.g., by enzymatic activity or cellular machinery, in somecases, for example, through exposure to a lysozyme (e.g., havingrelatively low pH). In some cases, the polymers may be broken down intomonomers and/or other nonpolymeric moieties that cells can either reuseor dispose of without significant toxic effect on the cells (forexample, polylactide may be hydrolyzed to form lactic acid,polyglycolide may be hydrolyzed to form glycolic acid, etc.).

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEGylated polymers andcopolymers of lactide and glycolide (e.g., PEGylated PLA, PEGylated PGA,PEGylated PLGA, and derivatives thereof. In some embodiments, polyestersinclude, for example, polyanhydrides, poly(ortho ester) PEGylatedpoly(ortho ester), poly(caprolactone), PEGylated poly(caprolactone),polylysine, PEGylated polylysine, poly(ethylene inline), PEGylatedpoly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester), poly[a-(4-aminobutyl)-L-glycolic acid],and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA are characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid-.glycolic acid ratio. In some embodiments, PLGA to be usedin accordance with the present invention is characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In particular embodiments, by optimizing the ratio of lactic acid toglycolic acid monomers in the polymer of the nanoparticle (e.g., thePLGA block copolymer or PLGA-PEG block copolymer), nanoparticleparameters such as water uptake, therapeutic agent release (e.g.,“controlled release”) and polymer degradation kinetics can be optimized.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid polyacrylamide, aminoalkyl methacrylate copolymer,glycidyl methacrylate copolymers, polycyanoacrylates, and combinationscomprising one or more of the foregoing polymers. The acrylic polymermay comprise fully-polymerized copolymers of acrylic and methacrylicacid esters with a low content of quaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g. DNA, RNA, or derivatives thereof).Amine-containing polymers such as poly(lysine) (Zauner et al., 1998,Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, BioconjugateChem., 6:7), polyethylene imine) (PEI; Boussif et al, 1995, Proc. Natl.Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers(Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897;Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,Bioconjugate Chem., 4:372) are positively-charged at physiological pH,form ion pairs with nucleic acids, and mediate transfection in a varietyof cell lines.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwonef al, 1999,Macromolecules, 22325Q-, Urn et al., 1999, J. Am. Chem. Soc., 121:5633;and Zhou et al, 1990, Macromolecules, 23:3399). Examples of thesepolyesters include poly(L-lactide-co-L-lysine) (Barrera et al, 1993, J.Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al, 1990,Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam et al,1999, Macromolecules, 32:3658; and Lim et al, 1999, J. Am. Chem. Soc.,121:5633). Poly(4-hydroxy-L-proline ester) was demonstrated to condenseplasmid DNA through electrostatic interactions, and to mediate genetransfer (Putnam et al, 1999, Macromolecules, 32:3658; and Lim et al,1999, J. Am. Chem. Soc., 121:5633). These new polymers are less toxicthan poly(lysine) and PEI, and they degrade into non-toxic metabolites.

A polymer (e.g., copolymer, e.g., block copolymer) containingpoly(ethylene glycol) repeat units is also referred to as a “PEGylated”polymer. Such polymers can control inflammation and/or immunogenicity(i.e., the ability to provoke an immune response) and/or lower the rateof clearance from the circulatory system via the reticuloendothelialsystem (RES), due to the presence of the poly(ethylene glycol) groups.

PEGylation may also be used, in some cases, to decrease chargeinteraction between a polymer and a biological moiety, e.g., by creatinga hydrophilic layer on the surface of the polymer, which may shield thepolymer from interacting with the biological moiety. In some cases, theaddition of poly(ethylene glycol) repeat units may increase plasmahalf-life of the polymer (e.g., copolymer, e.g., block copolymer), forinstance, by decreasing the uptake of the polymer by the phagocyticsystem while decreasing transfection/uptake efficiency by cells. Thoseof ordinary skill in the art will know of methods and techniques forPEGylating a polymer, for example, by using EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS(N-hydroxysuccinimide) to react a polymer to a PEG group terminating inan amine, by ring opening polymerization techniques (ROMP), or the like.

In another embodiment, the nanoparticle of the invention does notcontain PEG.

In addition, certain embodiments of the invention are directed towardscopolymers containing poly(ester-ether)s, e.g., polymers having repeatunits joined by ester bonds (e.g., R—C(O)—O—R′ bonds) and ether bonds(e.g., R—O—R′ bonds). In some embodiments of the invention, abiodegradable polymer, such as a hydrolyzable polymer, containingcarboxylic acid groups, may be conjugated with poly(ethylene glycol)repeat units to form a poly(ester-ether).

In a particular embodiment, the molecular weight of the polymers of thenanoparticles of the invention are optimized for effective treatment ofcancer, e.g., breast cancer. For example, the molecular weight of thepolymer influences nanoparticle degradation rate (particularly when themolecular weight of a biodegradable polymer is adjusted), solubility,water uptake, and drug release kinetics (e.g. “controlled release”). Asa further example, the molecular weight of the polymer can be adjustedsuch that the nanoparticle biodegrades in the subject being treatedwithin a reasonable period of time (ranging from a few hours to 1-2weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks, etc.). In particular embodimentsof a nanoparticle comprising a copolymer of PEG and PLGA, the PEG has amolecular weight of 1,000-20,000, e.g., 5,000-20,000, e.g.,10,000-20,000, and the PLGA has a molecular weight of 5,000-100,000,e.g., 20,000-70,000, e.g., 20,000-50,000.

In certain embodiments, the polymers of the nanoparticles may beconjugated to a lipid. The polymer may be, for example, alipid-terminated PEG. As described below, the lipid portion of thepolymer can be used for self assembly with another polymer, facilitatingthe formation of a nanoparticle. For example, a hydrophilic polymercould be conjugated to a lipid that will self assemble with ahydrophobic polymer.

In some embodiments, lipids are oils. In general, any oil known in theart can be conjugated to the polymers used in the invention. In someembodiments, an oil may comprise one or more fatty acid groups or saltsthereof. In some embodiments, a fatty acid group may comprisedigestible, long chain (e.g., C₈-C₅₀), substituted or unsubstitutedhydrocarbons. In some embodiments, a fatty acid group may be a C₁₀-C₂₀fatty acid or salt thereof. In some embodiments, a fatty acid group maybe a C₁₅-C₂₀ fatty acid or salt thereof. In some embodiments, a fattyacid may be unsaturated. In some embodiments, a fatty acid group may bemonounsaturated. In some embodiments, a fatty acid group may bepolyunsaturated. In some embodiments, a double bond of an unsaturatedfatty acid group may be in the cis conformation. In some embodiments, adouble bond of an unsaturated fatty acid may be in the transconformation.

In some embodiments, a fatty acid group may be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In a particular embodiment, the lipid is of the Formula V:

and salts thereof, wherein each R is, independently, C₁₋₃₀ alkyl. In oneembodiment of Formula V, the lipid is 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts thereof,e.g., the sodium salt.

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al,2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc.,123:2460; Langer, 2000, Ace. Chem. Res., 33:94; Langer, 1999, J.Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181).More generally, a variety of methods for synthesizing suitable polymersare described in Concise Encyclopedia of Polymer Science and PolymericAmines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980;Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al.,Prentice-Hall, 1981; Deming et al, 1997, Nature, 390:386; and in U.S.Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

In still another set of embodiments, a particle (comprising, e.g., acopolymer, e.g., a block copolymer) of the present invention includes atherapeutic moiety, i.e., a moiety that has a therapeutic orprophylactic effect when given to a subject. Examples of therapeuticmoieties to be used with the nanoparticles of the present inventioninclude antineoplastic or cytostatic agents or other agents withanticancer properties, or a combination thereof.

Thus, in certain embodiments, a library of such particles may becreated, as discussed herein.

In some cases, the particle is a nanoparticle, i.e., the particle has acharacteristic dimension of less than about 1 micrometer, where thecharacteristic dimension of a particle is the diameter of a perfectsphere having the same volume as the particle. For example, the particlemay have a characteristic dimension of the particle may be less thanabout 300 nm, less than about 200 nm, less than about 150 nm, less thanabout 100 nm, less than about 50 nm, less than about 30 nm, less thanabout 10 nm, less than about 3 nm, or less than about 1 nm in somecases. In particular embodiments, the nanoparticle of the presentinvention has a diameter of 50 nm-200 nm.

In some cases, a population of particles may be present. For example, apopulation of particles may include at least 20 particles, at least 50particles, at least 100 particles, at least 300 particles, at least1,000 particles, at least 3,000 particles, or at least 10,000 particles.Various embodiments of the present invention are directed to suchpopulations of particles. For instance, in some embodiments, theparticles may each be substantially the same shape and/or size(“monodisperse”). For example, the particles may have a distribution ofcharacteristic dimensions such that no more than about 5% or about 10%of the particles have a characteristic dimension greater than about 10%greater than the average characteristic dimension of the particles, andin some cases, such that no more than about 8%, about 5%, about 3%,about 1%, about 0.3%, about 0.1%, about 0.03%, or about 0.01% have acharacteristic dimension greater than about 10% greater man the averagecharacteristic dimension of the particles. In some cases, no more thanabout 5% of the particles have a characteristic dimension greater thanabout 5%, about 3%, about 1%, about 0.3%, about 0.1%, about 0.03%, orabout 0.01% greater than the average characteristic dimension of theparticles.

In one set of embodiments, the particles may have an interior and asurface, where the surface has a composition different from theinterior, i.e., there may be at least one compound present in theinterior but not present on the surface (or vice versa), and/or at leastone compound is present in the interior and on the surface at differingconcentrations. For example, in one embodiment, a compound, such as atargeting moiety (i.e., a poly(amino acid)) of a polymeric conjugate ofthe present invention, may be present in both the interior and thesurface of the particle, but at a higher concentration on the surfacethan in the interior of the particle, although in some cases, theconcentration in the interior of the particle may be essentiallynonzero, i.e., there is a detectable amount of the compound present inthe interior of the particle.

In some cases, the interior of the particle is more hydrophobic than thesurface of the particle. For instance, the interior of the particle maybe relatively hydrophobic with respect to the surface of the particle,and a drug or other pay load may be hydrophobic, and readily associateswith the relatively hydrophobic center of the particle. The drug orother payload may thus be contained within the interior of the particle,which may thus shelter it from the external environment surrounding theparticle (or vice versa). For instance, a drug or other payloadcontained within a particle administered to a subject will be protectedfrom a subject's body, and the body will also be isolated from the drug.A targeting moiety present on the surface of the particle may allow theparticle to become localized at a particular targeting site, forinstance, a tumor, a disease site, a tissue, an organ, a type of cell,etc. The drug or other payload may then, in some cases, be released fromthe particle and allowed to interact locally with the particulartargeting site. Yet another aspect of the invention is directed topolymer particles having more than one polymer or macromolecule present,and libraries involving such polymers or macromolecules. For example, inone set of embodiments, particles may contain more than onedistinguishable polymers (e.g., copolymers, e.g., block copolymers), andthe ratios of the two (or more) polymers may be independentlycontrolled, which allows for the control of properties of the particle.For instance, a first polymer may be a polymeric conjugate comprising atargeting moiety and a biocompatible portion, and a second polymer maycomprise a biocompatible portion but not contain the targeting moiety,or the second polymer may contain a distinguishable biocompatibleportion from the first polymer. Control of the amounts of these polymerswithin the polymeric particle may thus be used to control variousphysical, biological, or chemical properties of the particle, forinstance, the size of the particle (e.g., by varying the molecularweights of one or both polymers), the surface charge (e.g., bycontrolling the ratios of the polymers if the polymers have differentcharges or terminal groups), the surface hydrophilicity (e.g., if thepolymers have different molecular weights and/or hydrophilicities), thesurface density of the targeting moiety (e.g., by controlling the ratiosof the two or more polymers), etc.

As a specific example, a particle may comprise a first polymercomprising a poly(ethylene glycol) and a targeting moiety conjugated tothe poly(ethylene glycol), and a second polymer comprising thepoly(ethylene glycol) but not the targeting moiety, or comprising boththe poly(ethylene glycol) and the targeting moiety, where thepoly(ethylene glycol) of the second polymer has a different length (ornumber of repeat units) than the poly(ethylene glycol) of the firstpolymer. As another example, a particle may comprise a first polymercomprising a first biocompatible portion and a targeting moiety, and asecond polymer comprising a second biocompatible portion different fromthe first biocompatible portion (e.g., having a different composition, asubstantially different number of repeat units, etc.) and the targetingmoiety. As yet another example, a first polymer may comprise abiocompatible portion and a first targeting moiety, and a second polymermay comprise a biocompatible portion and a second targeting moietydifferent from the first targeting moiety.

Libraries of such particles may also be formed. For example, by varyingthe ratios of the two (or more) polymers within the particle, librariesof particles may be formed, which may be useful, for example, forscreening tests, high-throughput assays, or the like. Entities withinthe library may vary by properties such as those described above, and insome cases, more than one property of the particles may be varied withinthe library. Accordingly, one embodiment of the invention is directed toa library of nanoparticles having different ratios of polymers withdiffering properties. The library may include any suitable ratio(s) ofthe polymers.

In another embodiment, the nanoparticle is associated with (e.g.,surrounded by) a small molecule amphiphilic compound, giving the“amphiphilic-nanoparticle” three main components: 1) a biodegradablepolymeric material that forms the core of the particle, which can carrybioactive drugs and release them at a sustained rate after cutaneous,subcutaneous, mucosal, intramuscular, ocular, systemic, oral orpulmonary administration; 2) a small molecule amphiphilic compound thatsurrounds the polymeric material forming a shell for the particle; and3) a stealth material that can allow the particles to evade recognitionby immune system components and increase particle circulation half life.This embodiment may also include a fourth component: 4) a targetingmolecule that can bind to a unique molecular signature on cells,tissues, or organs of the body. In a preferred embodiment, theseparticles would be useful in drug delivery for therapeutic applications.In an alternative preferred embodiment, these particles would be usefulfor molecular imaging, for diagnostic applications, or for a combinationthereof.

In another embodiment, the amphiphilic-nanoparticle of the inventioncomprises a: 1) a biodegradable polymeric core which can carry bioactivedrugs and release them at a sustained rate; 2) a lipid monolayer shellwhich can prevent the carried agents from freely diffusing out of thenanoparticle and reduce water penetration rate into the nanoparticle,thereby enhancing drug encapsulation efficiency and slowing drugrelease; 3) a stealth material that can allow the particles to evaderecognition by immune system components and increase particlecirculation half life; and 4) a targeting molecule that can bind to aunique molecular signature on cells, tissues, or organs of the body.

In a preferred embodiment of the amphiphilic-nanoparticle, a poly(aminoacid) targeting molecule is first chemically conjugated to thehydrophilic region of a small molecule amphiphilic compound. Thisconjugate is then mixed with a certain ratio of unconjugated smallmolecule amphiphilic compounds in an aqueous solution containing one ormore water-miscible solvents. In a preferred embodiment, the poly(aminoacid) targeting molecule is one or a plurality of antibodies, aptamers,peptides, small molecules, or combinations thereof. The amphiphiliccompound can be, but is not limited to, one or a plurality of thefollowing: naturally derived lipids, surfactants, or synthesizedcompounds with both hydrophilic and hydrophobic moieties. The watermiscible solvent can be, but is not limited to: acetone, ethanol,methanol, and isopropyl alcohol. Separately, a biodegradable polymericmaterial is mixed with the agent or agents to be encapsulated in a watermiscible or partially water miscible organic solvent. In a preferredembodiment, the biodegradable polymer can be any of the biodegradablepolymers disclosed herein, for example, poly(D,L-lactic acid),poly(D,L-glycolic acid), poly(ε-caprolactone), or their copolymers atvarious molar ratios. The carried agent can be, but is not limited to,one or a plurality of the following therapeutic agents discussed below,including, for example, therapeutic drugs, imaging probes, orhydrophobic or lipophobic molecules for medical use. The water miscibleorganic solvent can be but is not limited to: acetone, ethanol,methanol, or isopropyl alcohol. The partially water miscible organicsolvent can be, but is not limited to: acetonitrile, tetrahydrofuran,ethyl acetate, isopropyl alcohol, isopropyl acetate, ordimethylformamide. The resulting polymer solution is then added to theaqueous solution of conjugated and unconjugated amphiphilic compound toyield nanoparticles by the rapid diffusion of the organic solvent intothe water and evaporation of the organic solvent.

As used herein, the term “amphiphilic” refers to a property where amolecule has both a polar portion and a non-polar portion. Often, anamphiphilic compound has a polar head attached to a long hydrophobictail. In some embodiments, the polar portion is soluble in water, whilethe non-polar portion is insoluble in water. In addition, the polarportion may have either a formal positive charge, or a formal negativecharge. Alternatively, the polar portion may have both a formal positiveand a negative charge, and be a zwitterion or inner salt. For purposesof the invention, the amphiphilic compound can be, but is not limitedto, one or a plurality of the following: naturally derived lipids,surfactants, or synthesized compounds with both hydrophilic andhydrophobic moieties.

Specific examples of amphiphilic compounds include, but are not limitedto, phospholipids, such as 1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), diarachidoylphosphatidylcholine (DAPC),dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine(DTPC), and dilignoceroylphatidylcholine (DLPC), incorporated at a ratioof between 0.01-60 (weight lipid/w polymer), most preferably between0.1-30 (weight lipid/w polymer). Phospholipids which may be usedinclude, but are not limited to, phosphatidic acids, phosphatidylcholines with both saturated and unsaturated lipids, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines,phosphatidylinositols, lysophosphatidyl derivatives, cardiolipin, andβ-acyl-y-alkyl phospholipids. Examples of phospholipids include, but arenot limited to, phosphatidylcholines such asdioleoylphosphatidylcholine, dimyristoylphosphatidylcholine,dipentadecanoylphosphatidylcholine dilauroylphosphatidylcholine,dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), diarachidoylphosphatidylcholine (DAPC),dibehenoylphosphatidylcho-line (DBPC), ditricosanoylphosphatidylcholine(DTPC), dilignoceroylphatidylcholine (DLPC); andphosphatidylethanolamines such as dioleoylphosphatidylethanolamine or1-hexadecyl-2-palmitoylglycerophos-phoethanolamine. Syntheticphospholipids with asymmetric acyl chains (e.g., with one acyl chain of6 carbons and another acyl chain of 12 carbons) may also be used.

In a particular embodiment, an amphiphilic component that can be used toform an amphiphilic layer is lecithin, and, in particular,phosphatidylcholine. Lecithin is an amphiphilic lipid and, as such,forms a phospholipid bilayer having the hydrophilic (polar) heads facingtheir surroundings, which are oftentimes aqueous, and the hydrophobictails facing each other. Lecithin has an advantage of being a naturallipid that is available from, e.g., soybean, and already has FDAapproval for use in other delivery devices. In addition, a mixture oflipids such as lethicin is more advantageous than one single pure lipid.

In certain embodiments of the invention, the amphiphilic layer of thenanoparticle, e.g., the layer of lecithin, is a monolayer, meaning thelayer is not a phospholipid bilayer, but exists as a single continuousor discontinuous layer around, or within, the nanoparticle. A monolayerhas the advantage of allowing the nanoparticles to be smaller in size,which makes them easier to prepare. The amphiphilic layer is “associatedwith” the nanoparticle of the invention, meaning it is positioned insome proximity to the polymeric matrix, such as surrounding the outsideof the polymeric matrix (e.g., PLGA), or dispersed within the polymersthat make up the nanoparticle.

By covering the polymeric nanoparticles with a thin film of smallmolecule amphiphilic compounds and conjugating poly(amino acid)targeting molecules to the amphiphilic compounds before formulatingnanoparticles, the disclosed invention has merits of both polymer- andlipid-based nanoparticles, while excluding some of their limitations.The amphiphilic compounds form a tightly assembled monolayer around thepolymeric core. This monolayer effectively prevents the carried agentsfrom freely diffusing out of the nanoparticle, thereby enhancing theencapsulation yield and slowing drug release. Moreover, the amphiphilicmonolayer also reduces water penetration rate into the nanoparticle,which slows hydrolysis rate of the biodegradable polymers, therebyincreasing particle stability and lifetime. In addition, by conjugatingtargeting ligands to the amphiphilic component prior to incorporatingthem into the nanoparticle, the composition of the nanoparticle and itssurface properties can be more accurately quantified.

In one embodiment, upon being administered to a subject, the amphipiliclayer of the nanoparticle of the invention can degrade, such that thepolymer core is eventually “unshielded.” Such a process, particularlywhen occurring after penetration into target tissue, can lead to moreefficient delivery of the therapeutic agent, thereby affording anenhanced therapeutic effect. Without being bound by theory, in the caseof basement membrane targeting, the nanoparticle may aggregate at thefirst collagen IV at the surface of the basement membrane. By “shedding”the lipid shell, the drug/polymer core can more deeply penetrate intothe basement membrane,

The surface of the nanoparticles of the invention can also be modifiedto enhance their arterial uptake. Nanoparticle surface modifying agentsinclude, but are not limited to, heparin, L-R-phosphatidylethanolamine,cyanoacrylate, epoxide, fibronectin, fibrinogen, ferritin, lipofectin,didodecyldimethylammonium bromide, and DEAE-Dextran, and any othersurface modifying agent disclosed in J Pharm Sci. 1998 October;87(10):1229-34, which is incorporated herein by reference in itentirety. The nanoparticulate system of the invention can also bemanipulated to have better compatibility with a drug delivery device,e.g., stent. For example, viscosity can be adjusted to adjust the dragforce of the nanoparticulate system.

In general, the nanoparticles of the present invention are about 40 nmto about 500 nm in size. In one embodiment, the nanoparticles of theinvention are less than or equal to about 90 nm in size, e.g., about 40nm to about 80 nm, e.g., about 40 nm to about 60 nm. Because thenanoparticles of the invention can be less than 90 nm in size, liveruptake by the subject is reduced, thereby allowing longer circulation inthe bloodstream.

In one embodiment, a nanoparticle of this invention is between 40 nm and80 nm in diameter and contains an amphiphilic component to polymerration of between 14:1 to 34:1. In one embodiment, a nanoparticle willhave approximately 10% to 40% lipid (by weight). In another embodiment,the nanoparticle will have a size of about 90 nm to about 40 nm. In oneembodiment, a nanoparticle that is approximately 10% to 40% lipid (byweight) will have a corresponding size of about 90 nm to about 40 nm.

The nanoparticles of the invention also have a surface zeta potentialranging from about −80 mV to 50 mV. Zeta potential is a measurement ofsurface potential of a particle. In some embodiments, the particles havea zeta potential ranging between 0 mV and −50 mV, e.g., between −1 mVand −50 mV. In some embodiments, the particles have a zeta potentialranging between −1 mV and −25 mV. In some embodiments, the particleshave a zeta potential ranging between −1.1 mV and −10 mV.

In other embodiments, the nanoparticles of the invention are liposomes,liposome polymer combinations, dendrimers, and albumin particles thatare functionalized with a poly(amino acid) ligand. These nanoparticlescan be used to deliver a therapeutic agent to a subject, such as ananti-cancer agent like mitoxantrone or docetaxel.

As used herein, the term “liposome” refers to a generally sphericalvesicle or capsid generally comprised of amphipathic molecules (e.g.,having both a hydrophobic (nonpolar) portion and a hydrophilic (polar)portion). Typically, the liposome can be produced as a single(unilamellar) closed bilayer or a multicompartment (multilamellar)closed bilayer. The liposome can be formed by natural lipids, syntheticlipids, or a combination thereof. In a preferred embodiment, theliposome comprises one or more phospholipids. Lipids known in the artfor forming liposomes include, but are not limited to, lecithin (soy oregg; phosphatidylcholine), dipalmitoylphosphatidylcholine,dimyristoylphosphatidylcholine, distearoylphosphatidylcholine,dicetylphosphate, phosphatidylglycerol, hydrogenatedphosphatidylcholine, phosphatidic acid, cholesterol,phosphatidylinositol, a glycolipid, phosphatidylethanolamine,phosphatidylserine, a maleimidyl-derivatized phospholipid (e.g.,N-[4(p-malei-midophenyl)butyryl] phosphatidylethanolamine),dioleylphosphatidylcholine, dipalmitoylphosphatidylglycerol,dimyristoylphosphatidic acid, and a combination thereof. Liposomes havebeen used to deliver therapeutic agents to cells.

The nanoparticles of the invention can also be “stealth liposomes,”which comprise lipids wherein the head group is modified with PEG. Thisresults in extended circulating half life in the subject.

Dendritic polymers (otherwise known as “dendrimers”) are uniformpolymers, variously referred to in the literature as hyperbrancheddendrimers, arborols, fractal polymers and starburst dendrimers, havinga central core, an interior dendritic (hyperbranched) structure and anexterior surface with end groups. These polymers differ from theclassical linear polymers both in form and function. Dendrimer chemistryconstructs macromolecules with tight control of size, shape topology,flexibility and surface groups (e.g., a poly(amino acid) ligand). Inwhat is known as divergent synthesis, these macromolecules start byreacting an initiator core in high-yield iterative reaction sequences tobuild symmetrical branches radiating from the core with well-definedsurface groups. Alternatively, in what is known as convergent synthesis,dendritic wedges are constructed from the periphery inwards towards afocal point and then several dendritic wedges are coupled at the focalpoints with a polyfunctional core. Dendritic syntheses form concentriclayers, known as generations, with each generation doubling themolecular mass and the number of reactive groups at the branch ends sothat the end generation dendrimer is a highly pure, uniform monodispersemacromolecule that solubilizes readily over a range of conditions. Forthe reasons discussed below, dendrimer molecular weights range from 300to 700,000 daltons and the number of surface groups (e.g., reactivesites for coupling) range significantly.

“Albumin particles” (also referred to as “albumin microspheres”) havebeen reported as carriers of pharmacological or diagnostic agents (see,e.g., U.S. Pat. Nos. 5,439,686; 5,498,421; 5,560,933; 5,665,382;6,096,331; 6,506,405; 6,537,579; 6,749,868; and 6,753,006; all of whichare incorporated herein by reference). Microspheres of albumin have beenprepared by either heat denaturation or chemical crosslinking. Heatdenatured microspheres are produced from an emulsified mixture (e.g.,albumin, the agent to be incorporated, and a suitable oil) attemperatures between 100° C. and 150° C. The microspheres are thenwashed with a suitable solvent and stored. Leucuta et al. (InternationalJournal of Pharmaceutics 41:213-217 (1988)) describe the method ofpreparation of heat denatured microspheres.

Poly(Amino Acid) Targeting Moieties

In yet another set of embodiments, the nanoparticles of the presentinvention includes a poly(amino acid) targeting moiety, i.e., a moietyable to bind to or otherwise associate with a biological entity, forexample, a membrane component, a cell surface receptor, Her-2, thebasement membrane of a blood vessel, basement membrane proteins,collagen, collagen IV or the like. In the case of the instant invention,the targeting moiety is a poly(amino acid) ligand. The term “bind” or“binding,” as used herein, refers to the interaction between acorresponding pair of molecules or portions thereof that exhibit mutualaffinity or binding capacity, typically due to specific or non-specificbinding or interaction, including, but not limited to, biochemical,physiological, and/or chemical interactions. “Biological binding”defines a type of interaction that occurs between pairs of moleculesincluding proteins, nucleic acids, glycoproteins, carbohydrates,hormones, or the like. The term “binding partner” refers to a moleculethat can undergo binding with a particular molecule. “Specific binding”refers to molecules, such as polynucleotides, that are able to bind toor recognize a binding partner (or a limited number of binding partners)to a substantially higher degree than to other, similar biologicalentities. In one set of embodiments, the targeting moiety has anaffinity (as measured via a disassociation constant) of less than about1 micromolar, at least about 10 micromolar, or at least about 100micromolar.

The term “poly(amino acid)” as used herein, refers to a protein,affibody, peptide, or peptidomimetic containing natural and unnaturalamino acids, modified amino acids or protected amino acids. The agentsto be incorporated in the polymeric nanocarrier and delivered to atarget cell or tissue by a conjugate of the present invention may betherapeutic, diagnostic, prophylactic or prognostic agents. Any chemicalcompound to be administered to an individual may be delivered using theconjugates of the invention. The agent may be a small molecule,organometallic compound, radionuclides, nucleic acid, protein, peptide,polynucleotide, metal, an isotopically labeled chemical compound, drug,vaccine, immunological agent, etc.

The term “affibody” (see, e.g., U.S. Pat. No. 5,831,012, incorporatedherein by reference) refers to highly specific affinity proteins thatcan be designed to bind to any desired target molecule. These antibodymimics can be manufactured to have the desired properties (specificityand affinity), while also being highly robust to withstand a broad rangeof analytical conditions, including pH and elevated temperature. Thespecific binding properties that can be engineered into each proteinallow it to have very high specificity and the desired affinity for acorresponding target protein. A specific target protein will thus bindonly to its corresponding capture protein.

The present invention further provides a nanoparticle conjugated to apoly(amino acid) that selectively targets tumor vasculature andselectively binds collagen, such as non-helical collagen. In anotherembodiment, the invention provides a nanoparticle conjugated to apoly(amino acid) that selectively targets breast tumor vasculature andthat selectively binds collagen, e.g., collagen IV, e.g., denaturedcollagen IV or native collagen IV. In a one embodiment, the inventionprovides a nanoparticle conjugated to a poly(amino acid) thatselectively targets tumor vasculature and that selectively binds thealpha 2 chain of collagen IV.

In preferred embodiments, the poly(amino acid) targeting moiety targetstissue basement membrane, such as the basement membrane of a bloodvessel. A “basement membrane” refers to a thin membrane upon which isposed a single layer of cells. The basement membrane is made up ofproteins held together by type IV collagen. The epithelial cells areanchored with hemidesmosome to the basement membrane. The end resultresembles a layer of tiles attached to a thin sheet. As discussed below,in cases where the endothelium is disrupted (by disease or trauma), thebasement membrane may be exposed and accessible to particles.

A variety of poly(amino acids) that selectively target tumor vasculatureare useful targeting moieties for the nanoparticles of the invention.Such poly(amino acids) include, without limitation, targeting peptidesand peptidomimetics. In one embodiment, the targeting peptide orpeptidomimetic portion of the nanoparticle has a length of at most 200residues. In another embodiment, the targeting peptide or peptidomimeticportion of the nanoparticle has a length of at most 50 residues. In afurther embodiment, a nanoparticle of the invention contains a targetingpeptide or peptidomimetic that includes the amino acid sequence AKERC,CREKA, ARYLQKLN or AXYLZZLN, wherein X and Z are variable amino acids,or conservative variants or peptidomimetics thereof. In particularembodiments, the poly(amino acid) targeting moiety is a peptide thatincludes the amino acid sequence AKERC, CREKA, ARYLQKLN or AXYLZZLN,wherein X and Z are variable amino acids, and has a length of less than20, 50 or 100 residues. The CREKA peptide is known in the art, and isdescribed in U.S. Patent Application No. 2005/0048063, which isincorporated herein by reference in its entirety. The octapeptideAXYLZZLN is described in Dinkla et al., The Journal of BiologicalChemistry, Vol. 282, No. 26, pp. 18686-18693, which is incorporatedherein by reference in its entirety.

Moreover, the authors of The Journal of Biological Chemistry, Vol. 282,No. 26, pp. 18686-18693 describe a binding motif in streptococci thatforms an autoantigenic complex with human collagen IV. Accordingly, anypeptide, or conservative variants or peptidomimetics thereof, that bindsor forms a complex with collagen IV, or the basement membrane of a bloodvessel, can be used as a targeting moiety for the nanoparticles of theinvention.

In one embodiment, the targeting moiety is an isolated peptide orpeptidomimetic that has a length of less than 100 residues and includesthe amino acid sequence CREKA (Cys Arg Glu Lys Ala) or a peptidomimeticthereof. Such an isolated peptide- or peptidomimetic can have, forexample, a length of less than 50 residues or a length of less than 20residues. In particular embodiments, the invention provides a peptidethat includes the amino acid sequence CREKA and has a length of lessthan 20, 50 or 100 residues.

As used herein in reference to a specified amino acid sequence, a“conservative variant” is a sequence in which a first amino acid isreplaced by another amino acid or amino acid analog having at least onebiochemical property similar to that of the first amino acid; similarproperties include, for example, similar size, charge, hydrophobicity orhydrogen-bonding capacity.

The peptides and peptidomimetics of the invention to be used aspoly(amino acid) targeting moieties are provided in isolated form. Asused herein in reference to a peptide or peptidomimetic of theinvention, the term “isolated” means a peptide or peptidomimetic that isin a form that is relatively free from material such as contaminatingpolypeptides, lipids, nucleic acids and other cellular material thatnormally is associated with the peptide or peptidomimetic in a cell orthat is associated with the peptide or peptidomimetic in a library or ina crude preparation.

The peptides and peptidomimetics of the invention to be used aspoly(amino acid) targeting moieties, including the bifunctional,multivalent and targeting peptides and peptidomimetics discussed below,can have a variety of lengths. A peptide or peptidomimetic of theinvention can have, for example, a relatively short length of less thansix, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35 or 40 residues. Apeptide or peptidomimetic of the invention also can be useful in thecontext of a significantly longer sequence. In another embodiment, apeptide or peptidomimetic of the invention can have, for example, alength of up to 50, 100, 150, 200, 250, 300, 400, 500, 1000 or 2000residues. In particular embodiments, a peptide or peptidomimetic of theinvention has a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90,100 or 200 residues. In further embodiments, a peptide or peptidomimeticof the invention has a length of 5 to 200 residues, 5 to 100 residues, 5to 90 residues, 5 to 80 residues, 5 to 70 residues, 5 to 60 residues, 5to 50 residues, 5 to 40 residues, 5 to 30 residues, 5 to 20 residues, 5to 15 residues, 5 to 10 residues, 10 to 200 residues, 10 to 100residues, 10 to 90 residues, 10 to 80 residues, 10 to 70 residues, 10 to60 residues, 10 to 50 residues, 10 to 40 residues, 10 to 30 residues, 10to 20 residues, 20 to 200 residues, 20 to 100 residues, 20 to 90residues, 20 to 80 residues, 20 to 70 residues, 20 to 60 residues, 20 to50 residues, 20 to 40 residues or 20 to 30 residues. As used herein, theterm “residue” refers to an amino acid or amino acid analog.

As used herein, the term “peptide” is used broadly to mean peptides,proteins, fragments of proteins and the like. The term “peptidomimetic,”as used herein, means a peptide-like molecule that has the activity ofthe peptide upon which it is structurally based. Such peptidomimeticsinclude chemically modified peptides, peptide-like molecules containingnon-naturally occurring amino acids, and peptoids and have an activitysuch as selective targeting activity of the peptide upon which thepeptidomimetic is derived (see, for example, Goodman and Ro,Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry andDrug Discovery” Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages803-861).

In another embodiment, the poly(amino acid) targeting moiety targetsHer-2. In a particular embodiment, the poly(amino acid) targeting moietyis an affibody that is an anti-HER2 affibody.

A polymeric conjugate to be used in the preparation of a nanoparticle ofthe present invention may be formed using any suitable conjugationtechnique. For instance, two components such as a targeting moiety and abiocompatible polymer, a biocompatible polymer and a poly(ethyleneglycol), etc., may be conjugated together using techniques such asEDC-NHS chemistry (1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride and N-hydroxysuccinimide) or a reaction involving amaleimide or a carboxylic acid, which can be conjugated to one end of athiol, an amine, or a similarly functionalized polyether. Theconjugation of such polymers, for instance, the conjugation of apoly(ester) and a poly(ether) to form a poly(ester-ether), can beperformed in an organic solvent, such as, but not limited to,dichloromethane, acetonitrile, chloroform, dimethylformamide,tetrahydrofuran, acetone, or the like. Specific reaction conditions canbe determined by those of ordinary skill in the art using no more thanroutine experimentation.

In another set of embodiments, a conjugation reaction may be performedby reacting a polymer that comprises a carboxylic acid functional group(e.g., a poly(ester-ether) compound) with a polymer or other moiety(such as a targeting moiety) comprising an amine. For instance, atargeting moiety, such as a poly(amino-acid) ligand, may be reacted withan amine to form an amine-containing moiety, which can then beconjugated to the carboxylic acid of the polymer. Such a reaction mayoccur as a single-step reaction, i.e., the conjugation is performedwithout using intermediates such as N-hydroxysuccinimide or a maleimide.The conjugation reaction between the amine-containing moiety and thecarboxylic acid-terminated polymer (such as a poly(ester-ether)compound) may be achieved, in one set of embodiments, by adding theamine-containing moiety, solubilized in an organic solvent such as (butnot limited to) dichloromethane, acetonitrile, chloroform,tetrahydrofuran, acetone, formamide, dimethylformamide, pyridines,dioxane, or dimethysulfoxide, to a solution containing the carboxylicacid-terminated polymer. The carboxylic acid-terminated polymer may becontained within an organic solvent such as, but not limited to,dichloromethane, acetonitrile, chloroform, dimethylformamide,tetrahydrofuran, or acetone. Reaction between the amine-containingmoiety and the carboxylic acid-terminated polymer may occurspontaneously, in some cases. Unconjugated reactants may be washed awayafter such reactions, and the polymer may be precipitated in solventssuch as, for instance, ethyl ether, hexane, methanol, or ethanol.

As a specific example, a poly(amino acid) ligand may be prepared as atargeting moiety in a particle as follows. Carboxylic acid modifiedpoly(lactide-co-glycolide) (PLGA-COOH) may be conjugated to anamine-modified heterobifunctional poly(ethylene glycol) (NH₂—PEG-COOH)to form a copolymer of PLGA-PEG-COOH. By using an amine-containingpoly(amino acid) ligand (NH₂-Lig), a triblock polymer of PLGA-PEG-Ligmay be formed by conjugating the carboxylic acid end of the PEG to theamine functional group on the ligand. The multiblock polymer can then beused, for instance, as discussed below, e.g., for therapeuticapplications.

Another aspect of the invention is directed to particles that includepolymer conjugates such as the ones described above. The particles mayhave a substantially spherical (i.e., the particles generally appear tobe spherical), or non-spherical configuration. For instance, theparticles, upon swelling or shrinkage, may adopt a non-sphericalconfiguration. In some cases, the particles may include polymericblends. For instance, a polymer blend may be formed that includes afirst polymer comprising a targeting moiety (i.e., a poly(amino acid)ligand) and a biocompatible polymer, and a second polymer comprising abiocompatible polymer but not comprising the targeting moiety. Bycontrolling the ratio of the first and second polymers in the finalpolymer, the concentration and location of targeting moiety in the finalpolymer may be readily controlled to any suitable degree.

Accordingly, the present invention provides poly(amino acid) targetingmoieties bound to a polymer. For example, the invention provides CREKAbound to PEG (CREKA-PEG), CREKA bound to PEG that is bound to a lipid(e.g., CREKA-PEG-DSPE), and CREKA bound to PEG-PLGA (CREKA-PEG-PLGA).The invention also provides the following conjugates:

wherein n is 20 to 1720; and

wherein R₇ is an alkyl group, R₈ is an ester or amide linkage, X=0 to 1mole fraction, Y=0 to 0.5 mole fraction, X+Y=20 to 1720, and Z=25 to455.

Preparation of Target-Specific Stealth Nanoparticles

Another aspect of the invention is directed to systems and methods ofproducing such target-specific stealth nanoparticles. In someembodiments, a solution containing a polymer is contacted with a liquid,such as an immiscible liquid, to form nanoparticles containing thepolymeric conjugate. Other aspects of the invention are directed tomethods of using such libraries, methods of using or administering suchpolymeric conjugates, methods of promoting the use of such polymericconjugates, kits involving such polymeric conjugates, or the like.

As mentioned, one aspect of the invention is directed to a method ofdeveloping nanoparticles with desired properties, such as desiredchemical, biological, or physical properties. In one set of embodiments,the method includes producing libraries of nanoparticles having highlycontrolled properties, which can be formed by mixing together two ormore polymers in different ratios. By mixing together two or moredifferent polymers (e.g., copolymers, e.g., block copolymers) indifferent ratios and producing particles from the polymers (e.g.,copolymers, e.g., block copolymers), particles having highly controlledproperties may be formed. For example, one polymer (e.g., copolymers,e.g., block copolymers) may include a poly(amino acid) ligand, whileanother polymer (e.g., copolymers, e.g., block copolymers) may be chosenfor its biocompatibility and/or its ability to control immunogenicity ofthe resultant particle.

Another aspect of the invention is directed to systems and methods ofmaking such particles. In one set of embodiments, the particles areformed by providing a solution comprising one or more polymers, andcontacting the solution with a polymer nonsolvent to produce theparticle. The solution may be miscible or immiscible with the polymernonsolvent. For example, a water-miscible liquid such as acetonitrilemay contain the polymers, and particles are formed as the acetonitrileis contacted with water, a polymer nonsolvent, e.g., by pouring theacetonitrile into the water at a controlled rate. The polymer containedwithin the solution, upon contact with the polymer nonsolvent, may thenprecipitate to form particles such as nanoparticles. Two liquids aresaid to be “immiscible” or not miscible, with each other when one is notsoluble in the other to a level of at least 10% by weight at ambienttemperature and pressure. Typically, an organic solution (e.g.,dichloromethane, acetonitrile, chloroform, tetrahydrofuran, acetone,formamide, dimethylformamide, pyridines, dioxane, dimethysulfoxide,etc.) and an aqueous liquid (e.g., water, or water containing dissolvedsalts or other species, cell or biological media, ethanol, etc.) areimmiscible with respect to each other. For example, the first solutionmay be poured into the second solution (at a suitable rate or speed). Insome cases, particles such as nanoparticles may be formed as the firstsolution contacts the immiscible second liquid, e.g., precipitation ofthe polymer upon contact causes the polymer to form nanoparticles whilethe first solution poured into the second liquid, and in some cases, forexample, when the rate of introduction is carefully controlled and keptat a relatively slow rate, nanoparticles may form. The control of suchparticle formation can be readily optimized by one of ordinary skill inthe art using only routine experimentation.

By creating a library of such particles, particles having any desirableproperties may be identified. For example, properties such as surfacefunctionality, surface charge, size, zeta (ζ) potential, hydrophobicity,ability to control immunogenicity, and the like, may be highlycontrolled. For instance, a library of particles may be synthesized, andscreened to identify the particles having a particular ratio of polymersthat allows the particles to have a specific density of moieties (e.g.,poly(amino acid) ligands) present on the surface of the particle. Thisallows particles having one or more specific properties to be prepared,for example, a specific size and a specific surface density of moieties,without an undue degree of effort. Accordingly, certain embodiments ofthe invention are directed to screening techniques using such libraries,as well as any particles identified using such libraries. In addition,identification may occur by any suitable method. For instance, theidentification may be direct or indirect, or proceed quantitatively orqualitatively.

In some embodiments, already-formed nanoparticles are functionalizedwith a targeting moiety using procedures analogous to those describedfor producing ligand-functionalized polymeric conjugates. As a specific,non-limiting example, this embodiment is exemplified schematically inFIG. 1A. In this figure, a first copolymer (PLGA-PEG,poly(lactide-co-glycolide) and poly(ethylene glycol)) is mixed with atherapeutic agent to form particles. The particles are then associatedwith a poly(amino acid) ligand to form nanoparticles that can be usedfor the treatment of cancer. The particles can be associated withvarying amounts of poly(amino acid) ligands in order to control thepoly(amino acid) ligand surface density of the nanoparticle, therebyaltering the therapeutic characteristics of the nanoparticle.Furthermore, for example, by controlling parameters such as PLGAmolecular weight, the molecular weight of PEG, and the nanoparticlesurface charge, very precisely controlled particles may be obtainedusing this method of preparation.

As a specific, non-limiting example, another embodiment is shownschematically in FIG. 1B. In this figure, a first copolymer (PLGA-PEG,poly(lactide-co-glycolide) and poly(ethylene glycol)) is conjugated to apoly(amino acid) ligand (PAALig) to form a PLGA-PEG-PAALig polymer. Thisligand-bound polymer is mixed with a second, non-functionalized polymer(PLGA-PEG in this example) at varying ratios to form a series ofparticles having different properties, for example, different surfacedensities of PSMA ligand as shown in this example. For example, bycontrolling parameters such as PLGA molecular weight, the molecularweight of PEG, the PSMA ligand surface density, and the nanoparticlesurface charge, very precisely controlled particles may be obtainedusing this method of preparation. As shown in FIG. 1B, the resultingnanoparticle can also include a therapeutic agent.

In another embodiment, the invention provides a method of preparing astealth nanoparticle wherein the nanoparticle has a ratio ofligand-bound polymer to non-functionalized polymer effective for thetreatment of breast cancer, wherein the hydrophilic, ligand-boundpolymer is conjugated to a lipid that will self assemble with thehydrophobic polymer, such that the hydrophobic and hydrophilic polymersthat constitute the nanoparticle are not covalently bound.“Self-assembly” refers to a process of spontaneous assembly of a higherorder structure that relies on the natural attraction of the componentsof the higher order structure (e.g., molecules) for each other. Ittypically occurs through random movements of the molecules and formationof bonds based on size, shape, composition, or chemical properties. Forexample, such a method comprises providing a first polymer that isreacted with a lipid, to form a polymer/lipid conjugate. Thepolymer/lipid conjugate is then reacted with the poly(amino acid) ligandto prepare a ligand-bound polymer/lipid conjugate; and mixing theligand-bound polymer/lipid conjugate with a second, non-functionalizedpolymer, and the therapeutic agent; such that the stealth nanoparticleis formed. In certain embodiments, the first polymer is PEG, such that alipid-terminated PEG is formed. In one embodiment, the lipid is of theFormula V, e.g., 2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),and salts thereof, e.g., the sodium salt. The lipid-terminated PEG canthen, for example, be mixed with PLGA to form a nanoparticle.

More generally, the polymers chosen to be used to create the library ofparticles may be any of a wide variety of polymers, such as described indetail below. Generally, two, three, four, or more polymers are mixed,in a wide range of ratios (e.g., each ranging from 0% to 100%), to formparticles such as nanoparticles having different ratios of each of thepolymers. The two or more polymers may be distinguishable in somefashion, e.g., having different polymeric groups, having the samepolymeric groups but with different molecular weights, having somepolymeric groups in common but having others that are different (e.g.,one may have a polymeric group that the other does not have), having thesame polymeric groups but in different orders, etc. The library ofparticles may have any number of members, for example, the library mayhave 2, 3, 5, 10, 30, 100, 300, 1000, 3000, 10,000, 30,000, 100,000,etc. members, which can be identified in some fashion. In some cases,the library may exist contemporaneously; for example, the library may becontained in one or more microtiter plates, vials, etc., or in someembodiments, the library may have include members created at differenttimes.

The library of particles can then be screened in some fashion toidentify those particles having one or more desired properties, forexample, surface functionality, surface charge, size, zeta (ζ)potential, hydrophobicity, ability to control immunogenicity, and thelike. One or more of the macromolecules within the particles may includeone or more polymers chosen to be biocompatible or biodegradable, one ormore polymers chosen to reduce immunogenicity, and/or one or morepoly(amino acid) ligands. These are discussed in detail below. Themacromolecules within the library may comprise some or all of thesepolymers, in any suitable combination (including, but not limited to,combinations in which a first polymer comprises a poly(amino acid)ligand and a second polymer does not contain any of these species).

As a specific example, in one embodiment, the particles may include afirst macromolecule comprising a biocompatible polymer, and a poly(aminoacid) ligand, and a second macromolecule comprising a biocompatiblepolymer, which may or may not be the same as that of the firstmacromolecule. As another example, a first macromolecule may be a blockcopolymer comprising a biocompatible hydrophobic polymer, abiocompatible hydrophilic polymer, and a poly(amino acid) ligand; and asecond macromolecule distinguishable from the first macromolecule insome fashion. For instance, the second macromolecule may comprise thesame (or a different) biocompatible hydrophobic polymer and the same (ora different) biocompatible hydrophilic polymer, but a differentpoly(amino acid) ligand (or no ligand at all) than the firstmacromolecule.

The nanoparticle of the invention may also be comprised of, as anotherexample, a first macromolecule comprising a biocompatible hydrophobicpolymer, a biocompatible hydrophilic polymer, and a poly(amino acid)ligand, and a second macromolecule that is distinguishable from thefirst macromolecule. For instance, the second macromolecule may containnone of the polymers of the first macromolecule, the secondmacromolecule may contain one or more polymers of the firstmacromolecule and one or more polymers not present in the firstmacromolecule, the second macromolecule may lack one or more of thepolymers of the first macromolecule, the second macromolecule maycontain all of the polymers of the first macromolecule, but in adifferent order and/or with one or more of the polymers having differentmolecular weights, etc.

As yet another example, the first macromolecule may comprise abiocompatible hydrophobic polymer, a biocompatible hydrophilic polymer,and a poly(amino acid) ligand, and the second macromolecule may comprisethe biocompatible hydrophobic polymer and the biocompatible hydrophilicpolymer, and be distinguishable from the first macromolecule in somefashion. As still another example, the first macromolecule may comprisea biocompatible hydrophobic polymer and a biocompatible hydrophilicpolymer, and the second macromolecule may comprise the biocompatiblehydrophobic polymer and a poly(amino acid) ligand, where the secondmacromolecule is distinguishable from the first macromolecule in somefashion.

The nanoparticles described above may also contain therapeutic agents.Examples of therapeutic agents include, but are not limited to, achemotherapeutic agent, a radioactive agent, a nucleic acid-based agent,a lipid-based agent, a carbohydrate based agent, a natural smallmolecule, or a synthetic small molecule.

The polymers or macromolecules may then be formed into a particle, usingtechniques such as those discussed in detail below. The geometry formedby the particle from the polymer or macromolecule may depend on factorssuch as the polymers that form the particle.

FIG. 2 illustrates that libraries can be produced using polymers such asthose described above. For example, in FIG. 2, polymeric particlescomprising a first macromolecule comprising a biocompatible hydrophobicpolymer, a biocompatible hydrophilic polymer, and a poly(amino acid)ligand, and a second macromolecule that comprises a biocompatiblehydrophobic polymer and a biocompatible hydrophilic polymer may be usedto create a library of particles having different ratios of the firstand second macromolecules.

Such a library may be useful in achieving particles having any number ofdesirable properties, for instance properties such as surfacefunctionality, surface charge, size, zeta (ζ) potential, hydrophobicity,ability to control immunogenicity, or the like. In FIG. 2, differentratios of the first and second macromolecules (including ratios whereone of the macromolecules is absent) are combined to produce particlesthat form the basis of the library.

For instance, as shown in FIG. 2, as the amount of the firstmacromolecule is increased, relative to the second macromolecule, theamount of moiety (e.g., poly(amino acid) ligand) present on the surfaceof the particle may be increased. Thus, any suitable concentration ofmoiety on the surface may be achieved simply by controlling the ratio ofthe first and second macromolecules in the particles. Accordingly, sucha library of particles may be useful in selecting or identifyingparticles having a particular functionality.

As specific examples, in some embodiments of the present invention, thelibrary includes particles comprising polymeric conjugates of abiocompatible polymer and a poly(amino acid) ligand, as discussedherein. Referring now to FIG. 3, one such particle is shown as anon-limiting example. In this figure, a polymeric conjugate of theinvention is used to form a particle 10. The polymer forming particle 10includes a poly(amino acid) 15, present on the surface of the particle,and a biocompatible portion 17. In some cases, as shown here, targetingmoiety 15 may be conjugated to biocompatible portion 17. However, notall of biocompatible portion 17 is shown conjugated to targeting moiety15. For instance, in some cases, particles such as particle 10 may beformed using a first polymer comprising biocompatible portion 17 andpoly(amino acid) ligand 15, and a second polymer comprisingbiocompatible portion 17 but not targeting moiety 15. By controlling theratio of the first and second polymers, particles having differentproperties may be formed, and in some cases, libraries of such particlesmay be formed. In addition, contained within the center of particle 10is drug 12. In some cases, drug 12 may be contained within the particledue to hydrophobic effects. For instance, the interior of the particlemay be relatively hydrophobic with respect to the surface of theparticle, and the drug may be a hydrophobic drug that associates withthe relatively hydrophobic center of the particle. In one embodiment,the therapeutic agent is associated with the surface of, encapsulatedwithin, surrounded by, or dispersed throughout the nanoparticle. Inanother embodiment, the therapeutic agent is encapsulated within thehydrophobic core of the nanoparticle.

As a specific example, particle 10 may contain polymers including arelatively hydrophobic biocompatible polymer and a relativelyhydrophilic targeting moiety 15, such that, during particle formation, agreater concentration of the hydrophilic targeting moiety is exposed onthe surface and a greater concentration of the hydrophobic biocompatiblepolymer is present within the interior of the particle.

In some embodiments, the biocompatible polymer is a hydrophobic polymer.Non-limiting examples of biocompatible polymers include polylactide,polyglycolide, and/or poly(lactide-co-glycolide).

In some cases, the polymeric conjugate is part of a controlled releasesystem. A “controlled release system,” as used herein, is a polymercombined with an active agent or a drug or other payload, such as atherapeutic agent, a diagnostic agent, a prognostic, a prophylacticagent, etc., and the active agent is released from the controlledrelease system in a predesigned or controlled manner. For example, theactive agent may be released in a constant manner over a predeterminedperiod of time, the active agent may be released in a cyclic manner overa predetermined period of time, or an environmental condition orexternal event may trigger the release of the active agent. Thecontrolled release polymer system may include a polymer that isbiocompatible, and in some cases, the polymer is biodegradable.

Therapeutic Agents

Another aspect of the present invention is directed to a therapeutic“payload,” or a species (or more than one species) contained within aparticle, such as those described above. For instance, the targetingmoiety may target or cause the particle to become localized at specificportions within a subject, and the payload may be delivered to thoseportions. In a particular embodiment, the drug or other payload isreleased in a controlled release manner from the particle and allowed tointeract locally with the particular targeting site (e.g., a tumor). Theterm “controlled release” (and variants of that term) as used herein(e.g., in the context of “controlled-release system”) is generally meantto encompass release of a substance (e.g., a drug) at a selected site orotherwise controllable in rate, interval, and/or amount. Controlledrelease encompasses, but is not necessarily limited to, substantiallycontinuous delivery, patterned delivery (e.g., intermittent deliveryover a period of time that is interrupted by regular or irregular timeintervals), and delivery of a bolus of a selected substance (e.g., as apredetermined, discrete amount if a substance over a relatively shortperiod of time (e.g., a few seconds or minutes)).

For example, a targeting portion may cause the particles to becomelocalized to a tumor, a disease site, a tissue, an organ, a type ofcell, etc. within the body of a subject, depending on the targetingmoiety used. For example, a poly(amino acid) ligand may become localizedto Her-2, the basement membrane of a blood vessel, collagen, collagen IVor the like. The subject may be a human or non-human animal. Examples ofsubjects include, but are not limited to, a mammal such as a dog, a cat,a horse, a donkey, a rabbit, a cow, a pig, a sheep, a goat, a rat, amouse, a guinea pig, a hamster, a primate, a human or the like.

In one set of embodiments, the payload is a drug or a combination ofmore than one drug. Such particles may be useful, for example, inembodiments where a targeting moiety may be used to direct a particlecontaining a drug to a particular localized location within a subject,e.g., to allow localized delivery of the drug to occur. Exemplarytherapeutic agents include chemotherapeutic agents such as doxorubicin(adriamycin), gemcitabine (gemzar), daunorubicin, procarbazine,mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU),vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel(taxotere), aldesleukin, asparaginase, busulfan, carboplatin, paltinderivatives, cladribine, camptothecin,CPT-1,10-hydroxy-7-ethylcamptothecin (SN38), dacarbazine, S—Icapecitabine, ftorafur, 5′deoxyfluorouridine, UFT, eniluracil,deoxycytidine, 5-azacyto sine, 5-azadeoxycyto sine, allopurinol,2-chloroadeno sine, trimetrexate, aminopterin,methylene-10-deazaminopterin (MDAM), oxaplatin, picoplatin, tetraplatin,satraplatin, platinum-DACH, ormaplatin, CI-973, JM-216, and analogsthereof, epirubicin, etoposide phosphate, 9-aminocamptothecin,10,11-methylenedioxycamptothecin, karenitecin, 9-nitrocamptothecin,TAS103, vindesine, L-phenylalanine mustard, ifosphamidemefosphamide,perfosfamide, trophosphamide carmustine, semustine, epothilones A-E,tomudex, 6-mercaptopurine, 6-thioguanine, amsacrine, etoposidephosphate, karenitecin, acyclovir, valacyclovir, ganciclovir,amantadine, rimantadine, lamivudine, zidovudine, bevacizumab,trastuzumab, rituximab, 5-Fluorouracil, and combinations thereof.

Suitable non-genetic therapeutic agents for use in connection with thepresent invention may be selected, for example, from one or more of thefollowing: (a) anti-thrombotic agents such as heparin, heparinderivatives, urokinase, clopidogrel, and PPack (dextrophenylalanineproline arginine chloromethylketone); (b) anti-inflammatory agents suchas dexamethasone, prednisolone, corticosterone, budesonide, estrogen,sulfasalazine and mesalamine; (c)antineoplastic/antiproliferative/anti-miotic agents such as paclitaxel,5-fluorouracil, cisplatin, vinblastine, vincristine, epothilones,endostatin, angiostatin, angiopeptin, monoclonal antibodies capable ofblocking smooth muscle cell proliferation, and thymidine kinaseinhibitors; (d) anesthetic agents such as lidocaine, bupivacaine andropivacaine; (e) anti-coagulants such as D-Phe-Pro-Arg chloromethylketone, an RGD peptide-containing compound, heparin, hirudin,antithrombin compounds, platelet receptor antagonists, anti-thrombinantibodies, anti-platelet receptor antibodies, aspirin, prostaglandininhibitors, platelet inhibitors and tick antiplatelet peptides; (f)vascular cell growth promoters such as growth factors, transcriptionalactivators, and translational promotors; (g) vascular cell growthinhibitors such as growth factor inhibitors, growth factor receptorantagonists, transcriptional repressors, translational repressors,replication inhibitors, inhibitory antibodies, antibodies directedagainst growth factors, bifunctional molecules consisting of a growthfactor and a cytotoxin, bifunctional molecules consisting of an antibodyand a cytotoxin; (h) protein kinase and tyrosine kinase inhibitors(e.g., tyrphostins, genistein, quinoxalines); (i) prostacyclin analogs;(j) cholesterol-lowering agents; (k) angiopoietins; (l) antimicrobialagents such as triclosan, cephalosporins, aminoglycosides andnitrofurantoin; (m) cytotoxic agents, cytostatic agents and cellproliferation affectors; (n) vasodilating agents; (o) agents thatinterfere with endogenous vasoactive mechanisms; (p) inhibitors ofleukocyte recruitment, such as monoclonal antibodies; (q) cytokines; (r)hormones; (s) inhibitors of HSP 90 protein (i.e., Heat Shock Protein,which is a molecular chaperone or housekeeping protein and is needed forthe stability and function of other client proteins/signal transductionproteins responsible for growth and survival of cells) includinggeldanamycin, (t) smooth muscle relaxants such as alpha receptorantagonists (e.g., doxazosin, tamsulosin, terazosin, prazosin andalfuzosin), calcium channel blockers (e.g., verapimil, diltiazem,nifedipine, nicardipine, nimodipine and bepridil), beta receptoragonists (e.g., dobutamine and salmeterol), beta receptor antagonists(e.g., atenolol, metaprolol and butoxamine), angiotensin-II receptorantagonists (e.g., losartan, valsartan, irbesartan, candesartan,eprosartan and telmisartan), and antispasmodic/anticholinergic drugs(e.g., oxybutynin chloride, flavoxate, tolterodine, hyoscyamine sulfate,diclomine), (u) bARKct inhibitors, (v) phospholamban inhibitors, (w)Serca 2 gene/protein, (x) immune response modifiers includingaminoquizolines, for instance, imidazoquinolines such as resiquimod andimiquimod, (y) human apolioproteins (e.g., AI, AII, AIII, AIV, AV,etc.), (z) selective estrogen receptor modulators (SERMs) such asraloxifene, lasofoxifene, arzoxifene, miproxifene, ospemifene, PKS 3741,MF 101 and SR 16234, (aa) PPAR agonists such as rosiglitazone,pioglitazone, netoglitazone, fenofibrate, bexaotene, metaglidasen,rivoglitazone and tesaglitazar, (bb) prostaglandin E agonists such asalprostadil or ONO 8815Ly, (cc) thrombin receptor activating peptide(TRAP), (dd) vasopeptidase inhibitors including benazepril, fosinopril,lisinopril, quinapril, ramipril, imidapril, delapril, moexipril andspirapril, (ee) thymosin beta 4, and (ff) phospholipids includingphosphorylcholine, phosphatidylinositol and phosphatidylcholine.

Preferred non-genetic therapeutic agents include taxanes such aspaclitaxel (including particulate forms thereof, for instance,protein-bound paclitaxel particles such as albumin-bound paclitaxelnanoparticles, e.g., ABRAXANE), sirolimus, everolimus, tacrolimus,zotarolimus, Epo D, dexamethasone, estradiol, halofuginone, cilostazole,geldanamycin, ABT-578 (Abbott Laboratories), trapidil, liprostin,Actinomcin D, Resten-NG, Ap-17, abciximab, clopidogrel, Ridogrel,beta-blockers, bARKct inhibitors, phospholamban inhibitors, Serca 2gene/protein, imiquimod, human apolioproteins (e.g., AI-AV), growthfactors (e.g., VEGF-2), as well derivatives of the forgoing, amongothers.

Suitable genetic therapeutic agents for use in connection with thepresent invention include anti-sense DNA and RNA as well as DNA codingfor the various proteins (as well as the proteins themselves) and may beselected, for example, from one or more of the following: (a) anti-senseRNA, (b) tRNA or rRNA to replace defective or deficient endogenousmolecules, (c) angiogenic and other factors including growth factorssuch as acidic and basic fibroblast growth factors, vascular endothelialgrowth factor, endothelial mitogenic growth factors, epidermal growthfactor, transforming growth factor α and β, platelet-derived endothelialgrowth factor, platelet-derived growth factor, tumor necrosis factor α,hepatocyte growth factor and insulin-like growth factor, (d) cell cycleinhibitors including CD inhibitors, and (e) thymidine kinase (“TK”) andother agents useful for interfering with cell proliferation. Also ofinterest is DNA encoding for the family of bone morphogenic proteins(“BMP's”), including BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 (Vgr-1), BMP-7(OP-1), BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15,and BMP-16. Currently preferred BMP's are any of BMP-2, BMP-3, BMP-4,BMP-5, BMP-6 and BMP-7. These dimeric proteins can be provided ashomodimers, heterodimers, or combinations thereof, alone or togetherwith other molecules. Alternatively, or in addition, molecules capableof inducing an upstream or downstream effect of a BMP can be provided.Such molecules include any of the “hedgehog” proteins, or the DNA'sencoding them.

Vectors for delivery of genetic therapeutic agents include viral vectorssuch as adenoviruses, gutted adenoviruses, adeno-associated virus,retroviruses, alpha virus (Semliki Forest, Sindbis, etc.), lentiviruses,herpes simplex virus, replication competent viruses (e.g., ONYX-015) andhybrid vectors; and non-viral vectors such as artificial chromosomes andmini-chromosomes, plasmid DNA vectors (e.g., pCOR), cationic polymers(e.g., polyethyleneimine, polyethyleneimine (PEI)), graft copolymers(e.g., polyether-PEI and polyethylene oxide-PEI), neutral polymers suchas polyvinylpyrrolidone (PVP), SP1017 (SUPRATEK), lipids such ascationic lipids, liposomes, lipoplexes, nanoparticles, ormicroparticles, with and without targeting sequences such as the proteintransduction domain (PTD).

Cells for use in conjunction with the present invention include cells ofhuman origin (autologous or allogeneic), including whole bone marrow,bone marrow derived mono-nuclear cells, progenitor cells (e.g.,endothelial progenitor cells), stem cells (e.g., mesenchymal,hematopoietic, neuronal), pluripotent stem cells, fibroblasts,myoblasts, satellite cells, pericytes, cardiomyocytes, skeletal myocytesor macrophage, or from an animal, bacterial or fungal source(xenogeneic), which can be genetically engineered, if desired, todeliver proteins of interest.

Further therapeutic agents, not necessarily exclusive of those listedabove, have been identified as candidates for vascular treatmentregimens, for example, as agents targeting restenosis (anti-restenoticagents). Suitable agents may be selected, for example, from one or moreof the following: (a) Ca-channel blockers including benzothiazapinessuch as diltiazem and clentiazem, dihydropyridines such as nifedipine,amlodipine and nicardapine, and phenylalkylamines such as verapamil, (b)serotonin pathway modulators including: 5-HT antagonists such asketanserin and naftidrofuryl, as well as 5-HT uptake inhibitors such asfluoxetine, (c) cyclic nucleotide pathway agents includingphosphodiesterase inhibitors such as cilostazole and dipyridamole,adenylate/Guanylate cyclase stimulants such as forskolin, as well asadenosine analogs, (d) catecholamine modulators including α-antagonistssuch as prazosin and bunazosine, β-antagonists such as propranolol andα/β-antagonists such as labetalol and carvedilol, (e) endothelinreceptor antagonists such as bosentan, sitaxsentan sodium, atrasentan,endonentan, (f) nitric oxide donors/releasing molecules includingorganic nitrates/nitrites such as nitroglycerin, isosorbide dinitrateand amyl nitrite, inorganic nitroso compounds such as sodiumnitroprusside, sydnonimines such as molsidomine and linsidomine,nonoates such as diazenium diolates and NO adducts of alkanediamines,S-nitroso compounds including low molecular weight compounds (e.g.,S-nitroso derivatives of captopril, glutathione and N-acetylpenicillamine) and high molecular weight compounds (e.g., S-nitrosoderivatives of proteins, peptides, oligosaccharides, polysaccharides,synthetic polymers/oligomers and natural polymers/oligomers), as well asC-nitroso-compounds, O-nitroso-compounds, N-nitroso-compounds andL-arginine, (g) Angiotensin Converting Enzyme (ACE) inhibitors such ascilazapril, fosinopril and enalapril, (h) ATII-receptor antagonists suchas saralasin and losartin, (i) platelet adhesion inhibitors such asalbumin and polyethylene oxide, (j) platelet aggregation inhibitorsincluding cilostazole, aspirin and thienopyridine (ticlopidine,clopidogrel) and GP IIb/IIIa inhibitors such as abciximab, epitifibatideand tirofiban, (k) coagulation pathway modulators including heparinoidssuch as heparin, low molecular weight heparin, dextran sulfate andβ-cyclodextrin tetradecasulfate, thrombin inhibitors such as hirudin,hirulog, PPACK(D-phe-L-propyl-L-arg-chloromethylketone) and argatroban,FXa inhibitors such as antistatin and TAP (tick anticoagulant peptide),Vitamin K inhibitors such as warfarin, as well as activated protein C,(l) cyclooxygenase pathway inhibitors such as aspirin, ibuprofen,flurbiprofen, indomethacin and sulfinpyrazone, (m) natural and syntheticcorticosteroids such as dexamethasone, prednisolone, methprednisoloneand hydrocortisone, (n) lipoxygenase pathway inhibitors such asnordihydroguairetic acid and caffeic acid, (o) leukotriene receptorantagonists, (p) antagonists of E- and P-selectins, (q) inhibitors ofVCAM-1 and ICAM-1 interactions, (r) prostaglandins and analogs thereofincluding prostaglandins such as PGE1 and PGI2 and prostacyclin analogssuch as ciprostene, epoprostenol, carbacyclin, iloprost and beraprost,(s) macrophage activation preventers including bisphosphonates, (t)HMG-CoA reductase inhibitors such as lovastatin, pravastatin,atorvastatin, fluvastatin, simvastatin and cerivastatin, (u) fish oilsand omega-3-fatty acids, (v) free-radical scavengers/antioxidants suchas probucol, vitamins C and E, ebselen, trans-retinoic acid and SOD(orgotein), SOD mimics, verteporfin, rostaporfin, AGI 1067, and M 40419,(w) agents affecting various growth factors including FGF pathway agentssuch as bFGF antibodies and chimeric fusion proteins, PDGF receptorantagonists such as trapidil, IGF pathway agents including somatostatinanalogs such as angiopeptin and ocreotide, TGF-β pathway agents such aspolyanionic agents (heparin, fucoidin), decorin, and TGF-β antibodies,EGF pathway agents such as EGF antibodies, receptor antagonists andchimeric fusion proteins, TNF-α pathway agents such as thalidomide andanalogs thereof, Thromboxane A2 (TXA2) pathway modulators such assulotroban, vapiprost, dazoxiben and ridogrel, as well as proteintyrosine kinase inhibitors such as tyrphostin, genistein and quinoxalinederivatives, (x) matrix metalloprotease (MMP) pathway inhibitors such asmarimastat, ilomastat, metastat, batimastat, pentosan polysulfate,rebimastat, incyclinide, apratastat, PG 116800, RO 1130830 or ABT 518,(y) cell motility inhibitors such as cytochalasin B, (z)antiproliferative/antineoplastic agents including antimetabolites suchas purine analogs (e.g., 6-mercaptopurine or cladribine, which is achlorinated purine nucleoside analog), pyrimidine analogs (e.g.,cytarabine and 5-fluorouracil) and methotrexate, nitrogen mustards,alkyl sulfonates, ethylenimines, antibiotics (e.g., daunorubicin,doxorubicin), nitrosoureas, cisplatin, agents affecting microtubuledynamics (e.g., vinblastine, vincristine, colchicine, Epo D, paclitaxeland epothilone), caspase activators, proteasome inhibitors, angiogenesisinhibitors (e.g., endostatin, angiostatin and squalamine), rapamycin(sirolimus) and its analogs (e.g., everolimus, tacrolimus, zotarolimus,etc.), cerivastatin, flavopiridol and suramin, (aa) matrixdeposition/organization pathway inhibitors such as halofuginone or otherquinazolinone derivatives, pirfenidone and tranilast, (bb)endothelialization facilitators such as VEGF and RGD peptide, (cc) bloodrheology modulators such as pentoxifylline and (dd) glucose cross-linkbreakers such as alagebrium chloride (ALT-711).

Numerous additional therapeutics for the practice of the presentinvention may be selected from suitable therapeutic agents disclosed inU.S. Pat. No. 5,733,925 to Kunz.

Non-limiting examples of potentially suitable drugs include anti-canceragents, including, for example, docetaxel, mitoxantrone, andmitoxantrone hydrochloride. In another embodiment, the payload may be ananti-cancer drug such as 20-epi-1, 25 dihydroxyvitamin D3,4-ipomeanol,5-ethynyluracil, 9-dihydrotaxol, abiraterone, acivicin, aclarubicin,acodazole hydrochloride, acronine, acylfiilvene, adecypenol, adozelesin,aldesleukin, all-tk antagonists, altretamine, ambamustine, ambomycin,ametantrone acetate, amidox, amifostine, aminoglutethimide,aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole,andrographolide, angiogenesis inhibitors, antagonist D, antagonist G,antarelix, anthramycin, anti-dorsalizdng morphogenetic protein-1,antiestrogen, antineoplaston, antisense oligonucleotides, aphidicolinglycinate, apoptosis gene modulators, apoptosis regulators, apurinicacid, ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin,asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2,axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine, azetepa,azotomycin, baccatin III derivatives, balanol, batimastat,benzochlorins, benzodepa, benzoylstaurosporine, beta lactam derivatives,beta-alethine, betaclamycin B, betulinic acid, BFGF inhibitor,bicalutamide, bisantrene, bisantrene hydrochloride,bisazuidinylspermine, bisnafide, bisnafide dimesylate, bistratene A,bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists, breflate,brequinar sodium, bropirimine, budotitane, busulfan, buthioninesulfoximine, cactinomycin, calcipotriol, calphostin C, calusterone,camptothecin derivatives, canarypox IL-2, capecitabine, caraceraide,carbetimer, carboplatin, carboxamide-amino-triazole,carboxyamidotriazole, carest M3, carmustine, earn 700, cartilage derivedinhibitor, carubicin hydrochloride, carzelesin, casein kinaseinhibitors, castanosperrnine, cecropin B, cedefingol, cetrorelix,chlorambucil, chlorins, chloroquinoxaline sulfonamide, cicaprost,cirolemycin, cisplatin, cis-porphyrin, cladribine, clomifene analogs,clotrimazole, collismycin A, collismycin B, combretastatin A4,combretastatin analog, conagenin, crambescidin 816, crisnatol, crisnatolmesylate, cryptophycin 8, cryptophycin A derivatives, curacin A,cyclopentanthraquinones, cyclophosphamide, cycloplatam, cypemycin,cytarabine, cytarabine ocfosfate, cytolytic factor, cytostatin,dacarbazine, dacliximab, dactinomycin, daunorubicin hydrochloride,decitabine, dehydrodidemnin B, deslorelin, dexifosfamide, dexormaplatin,dexrazoxane, dexverapamil, dezaguanine, dezaguanine mesylate,diaziquone, didemnin B, didox, diethyhiorspermine,dihydro-5-azacytidine, dioxamycin, diphenyl spiromustine, docetaxel,docosanol, dolasetron, doxifluridine, doxorubicin, doxorubicinhydrochloride, droloxifene, droloxifene citrate, dromostanolonepropionate, dronabinol, duazomycin, duocannycin SA, ebselen, ecomustine,edatrexate, edelfosine, edrecolomab, eflomithine, eflomithinehydrochloride, elemene, elsarnitrucin, emitefur, enloplatin, enpromate,epipropidine, epirubicin, epirubicin hydrochloride, epristeride,erbulozole, erythrocyte gene therapy vector system, esorubicinhydrochloride, estramustine, estramustine analog, estramustine phosphatesodium, estrogen agonists, estrogen antagonists, etanidazole, etoposide,etoposide phosphate, etoprine, exemestane, fadrozole, fadrozolehydrochloride, fazarabine, fenretinide, filgrastim, finasteride,flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine,fludarabine phosphate, fluorodaunorunicin hydrochloride, fluorouracil,fluorocitabine, forfenimex, formestane, fosquidone, fostriecin,fostriecin sodium, fotemustine, gadolinium texaphyrin, gallium nitrate,galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, gemcitabinehydrochloride, glutathione inhibitors, hepsulfam, heregulin,hexamethylene bisacetamide, hydroxyurea, hypericin, ibandronic acid,idarubicin, idarubicin hydrochloride, idoxifene, idramantone,ifosfamide, ihnofosine, ilomastat, imidazoacridones, imiquimod,immunostimulant peptides, insulin-like growth factor-1 receptorinhibitor, interferon agonists, interferon alpha-2A, interferonalpha-2B, interferon alpha-N1, interferon alpha-N3, interferon beta-IA,interferon gamma-IB, interferons, interleukins, iobenguane,iododoxorubicin, iproplatm, irinotecan, irinotecan hydrochloride,iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron,jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide,lanreotide acetate, leinamycin, lenograstim, lentinan sulfate,leptolstatin, letrozole, leukemia inhibiting factor, leukocyte alphainterferon, leuprolide acetate, leuprolide/estrogen/progesterone,leuprorelin, levamisole, liarozole, liarozole hydrochloride, linearpolyamine analog, lipophilic disaccharide peptide, lipophilic platinumcompounds, lissoclinamide, lobaplatin, lombricine, lometrexol,lometrexol sodium, lomustine, lonidamine, losoxantrone, losoxantronehydrochloride, lovastatin, loxoribine, lurtotecan, lutetium texaphyrinlysofylline, lytic peptides, maitansine, mannostatin A, marimastat,masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinaseinhibitors, maytansine, mechlorethamine hydrochloride, megestrolacetate, melengestrol acetate, melphalan, menogaril, merbarone,mercaptopurine, meterelin, methioninase, methotrexate, methotrexatesodium, metoclopramide, metoprine, meturedepa, microalgal protein kinaseC uihibitors, MIF inhibitor, mifepristone, miltefosine, mirimostim,mismatched double stranded RNA, mitindomide, mitocarcin, mitocromin,mitogillin, mitoguazone, mitolactol, mitomalcin, mitomycin, mitomycinanalogs, mitonafide, mitosper, mitotane, mitotoxin fibroblast growthfactor-saporin, mitoxantrone, mitoxantrone hydrochloride, mofarotene,molgramostim, monoclonal antibody, human chorionic gonadotrophin,monophosphoryl lipid a/myobacterium cell wall SK, mopidamol, multipledrug resistance gene inhibitor, multiple tumor suppressor 1-basedtherapy, mustard anticancer agent, mycaperoxide B, mycobacterial cellwall extract, mycophenolic acid, myriaporone, n-acetyldinaline,nafarelin, nagrestip, naloxone/pentazocine, napavin, naphterpin,nartograstim, nedaplatin, nemorubicin, neridronic acid, neutralendopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxideantioxidant, nitrullyn, nocodazole, nogalamycin, n-substitutedbenzamides, O6-benzylguanine, octreotide, okicenone, oligonucleotides,onapristone, ondansetron, oracin, oral cytokine inducer, ormaplatin,osaterone, oxaliplatin, oxaunomycin, oxisuran, paclitaxel, paclitaxelanalogs, paclitaxel derivatives, palauamine, palmitoylrhizoxin,pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine,pegaspargase, peldesine, peliomycin, pentamustine, pentosan polysulfatesodium, pentostatin, pentrozole, peplomycin sulfate, perflubron,perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate,phosphatase inhibitors, picibanil, pilocarpine hydrochloride,pipobroman, piposulfan, pirarubicin, piritrexim, piroxantronehydrochloride, placetin A, placetin B, plasminogen activator inhibitor,platinum complex, platinum compounds, platinum-triamine complex,plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine,procarbazine hydrochloride, propyl bis-acridone, prostaglandin J2,prostatic carcinoma antiandrogen, proteasome inhibitors, protein A-basedimmune modulator, protein kinase C inhibitor, protein tyrosinephosphatase inhibitors, purine nucleoside phosphorylase inhibitors,puromycin, puromycin hydrochloride, purpurins, pyrazorurin,pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugate,RAF antagonists, raltitrexed, ramosetron, RAS farnesyl proteintransferase inhibitors, RAS inhibitors, RAS-GAP inhibitor, retelliptinedemethylated, rhenium RE 186 etidronate, rhizoxin, riboprine, ribozymes,RH retinamide, RNAi, rogletimide, rohitukine, romurtide, roquinimex,rubiginone B1, ruboxyl, safingol, safingol hydrochloride, saintopin,sarcnu, sarcophytol A, sargramostim, SDI1 mimetics, semustine,senescence derived inhibitor 1, sense oligonucleotides, signaltransduction inhibitors, signal transduction modulators, simtrazene,single chain antigen binding protein, sizofuran, sobuzoxane, sodiumborocaptate, sodium phenylacetate, solverol, somatomedin bindingprotein, sonermin, sparfosafe sodium, sparfosic acid, sparsomycin,spicamycin D, spirogermanium hydrochloride, spiromustine, spiroplatin,splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-celldivision inhibitors, stipiamide, streptonigrin, streptozocin,stromelysin inhibitors, sulfinosine, sulofenur, superactive vasoactiveintestinal peptide antagonist, suradista, suramin, swainsonine,synthetic glycosaminoglycans, talisomycin, tallimustine, tamoxifenmethiodide, tauromustine, tazarotene, tecogalan sodium, tegafur,tellurapyrylium, telomerase inhibitors, teloxantrone hydrochloride,temoporfin, temozolomide, teniposide, teroxirone, testolactone,tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide,thiamiprine, thiocoraline, thioguanine, thiotepa, thrombopoietin,thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist,thymotrinan, thyroid stimulating hormone, tiazofurin, tin ethyletiopurpurin, tirapazamine, titanocene dichloride, topotecanhydrochloride, topsentin, toremifene, toremifene citrate, totipotentstem cell factor, translation inhibitors, trestolone acetate, tretinoin,triacetyluridine, triciribine, triciribine phosphate, trimetrexate,trimetrexate glucuronate, triptorelin, tropisetron, tubulozolehydrochloride, turosteride, tyrosine kinase inhibitors, tyrphostins, UBCinhibitors, ubenimex, uracil mustard, uredepa, urogenital sinus-derivedgrowth inhibitory factor, urokinase receptor antagonists, vapreotide,variolin B, velaresol, veramine, verdins, verteporfin, vinblastinesulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidinesulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine,vinorelbine tartrate, vinrosidine sulfate, vinxaltine, vinzolidinesulfate, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb,zinostatin, zinostatin stimalamer, or zorubicin hydrochloride.

In another embodiment, the nanoparticles of the invention can be used totreat vulnerable plaque in a subject in need thereof. In particular, thepayload associated with, i.e., encapsulated within, the nanoparticle ofthe invention is a biologically active agent used to stabilize avulnerable plaque. Such agents are described in U.S. Pat. No. 7,008,411,which is incorporated herein by reference in its entirety.

In another embodiment, the nanoparticles of the invention can be used totreat restenosis or atherosclerosis in a subject in need thereof.Restenosis is the reobstruction of an artery following interventionalprocedures such as balloon angioplasty or stenting.

Additional examples of potentially suitable drugs include for deliveryby the nanoparticles of the invention include doxorubicin,2-aminochromone (U-86983, Upjohn and Pharmacia) (U-86), cytarabine,vincristine, dalteparin sodium, cyclosporine A, colchicines, etoposide,sirolimus, paclitaxel, ceramide, cilostazol, clodronate, pamidronate,alendronate, ISA-13-1, AG-1295, AGL-2043, dexamethasone, everolimus,ABT-578, tacrolimus (FK506), estradiol, lantrunculinD, cytochalasin A,dexamethasone, zotarolimus, angiopeptin, bisphosphonates, estrogen,angiopeptin, ROCK inhibitors, PDGF inhibitors, MMP inhibitors, statins,and well as combinations of these therapies (e.g., a combination ofzotarolimus and dexamethasone), as well as any therapeutic disclosed inCirc Res 2003 Apr. 18; 92(7):e62-9. Epub 2003 Mar. 27; J Pharm Sci 1998October; 87(10):1229-34; Int J Nanomedicine 2007; 2(2):143-61; andAtherosclerosis 2002 February; 160(2):259-71, which are incorporatedherein by reference in their entirety.

In one embodiment, therapeutic or biologically active agents may bereleased by the nanoparticles of the invention to induce therapeuticangiogenesis, which refers to the processes of causing or inducingangiogenesis and arteriogenesis, either downstream, or away from thevulnerable plaque. Arteriogenesis is the enlargement of pre-existingcollateral vessels. Collateral vessels allow blood to flow from awell-perfused region of the vessel into an ischemic region (from abovean occlusion to downstream from the occlusion). Angiogenesis is thepromotion or causation of the formation of new blood vessels downstreamfrom the ischemic region. Having more blood vessels (e.g., capillaries)below the occlusion may provide for less pressure drop to perfuse areaswith severe narrowing caused by a thrombus. In the event that anocclusive thrombus occurs in a vulnerable plaque, the myocardiumperfused by the affected artery is salvaged. Representative therapeuticor biologically active agents include, but are not limited to, proteinssuch as vascular endothelial growth factor (VEGF) in any of its multipleisoforms, fibroblast growth factors, monocyte chemoatractant protein 1(MCP-1), transforming growth factor alpha (TGF-alpha), transforminggrowth factor beta (TGF-beta) in any of its multiple isoforms, DEL-1,insulin like growth factors (IGF), placental growth factor (PLGF),hepatocyte growth factor (HGF), prostaglandin E1 (PG-E1), prostaglandinE2 (PG-E2), tumor necrosis factor alpha (THF-alpha), granulocytestimulating growth factor (G-CSF), granulocyte macrophagecolony-stimulating growth factor (GM-CSF), angiogenin, follistatin, andproliferin, genes encoding these proteins, cells transfected with thesegenes, pro-angiogenic peptides such as PR39 and PR11, and pro-angiogenicsmall molecules such as nicotine. The nanoparticles of the invention mayalso include lipid lowering agents (e.g., hydroxy-methylglutarylcoenzyme A (HMG CoA) reductase inhibitors, statins, niacin, bile acidresins, and fibrates), antioxidants (e.g., vitamin E (α-tocopherol),vitamin C, and β-carotene supplements), extracellular matrix synthesispromoters, inhibitors of plaque inflammation and extracellulardegradation, estradiol drug classes and its derivatives.

Other therapeutic agents to be delivered in accordance with the presentinvention include, but are not limited to, nucleic acids (e.g., siRNA,RNAi, and microRNA agents), proteins (e.g. antibodies), peptides,lipids, carbohydrates, hormones, metals, radioactive elements andcompounds, vaccines, immunological agents, etc., and/or combinationsthereof. In some embodiments, the agent to be delivered is an agentuseful in the treatment of cancer (e.g., prostate cancer).

In one embodiment, the nanoparticles of this invention will containnucleic acids such as siRNA. Preferably, the siRNA molecule has a lengthfrom about 10-50 or more nucleotides. More preferably, the siRNAmolecule has a length from about 15-45 nucleotides. Even morepreferably, the siRNA molecule has a length from about 19-40nucleotides. Even more preferably, the siRNA molecule has a length offrom about 21-23 nucleotides.

The siRNA of the invention preferably mediates RNAi against a targetmRNA. The siRNA molecule can be designed such that every residue iscomplementary to a residue in the target molecule. Alternatively, one ormore substitutions can be made within the molecule to increase stabilityand/or enhance processing activity of said molecule. Substitutions canbe made within the strand or can be made to residues at the ends of thestrand.

The target mRNA cleavage reaction guided by siRNAs is sequence specific.In general, siRNA containing a nucleotide sequence identical to aportion of the target gene are preferred for inhibition. However, 100%sequence identity between the siRNA and the target gene is not requiredto practice the present invention. Sequence variations can be toleratedincluding those that might be expected due to genetic mutation, strainpolymorphism, or evolutionary divergence. For example, siRNA sequenceswith insertions, deletions, and single point mutations relative to thetarget sequence have also been found to be effective for inhibition.Alternatively, siRNA sequences with nucleotide analog substitutions orinsertions can be effective for inhibition.

Moreover, not all positions of an siRNA contribute equally to targetrecognition. Mismatches in the center of the siRNA are most critical andessentially abolish target RNA cleavage. In contrast, the 3′ nucleotidesof the siRNA do not contribute significantly to specificity of thetarget recognition. Generally, residues at the 3′ end of the siRNAsequence which is complementary to the target RNA (e.g., the guidesequence) are not critical for target RNA cleavage.

Sequence identity may readily be determined by sequence comparison andalignment algorithms known in the art. To determine the percent identityof two nucleic acid sequences (or of two amino acid sequences), thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. NatL Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. NatL Acad. Sci. USA 90:5873. Such an algorithm isincorporated into the BLAST programs (version 2.0) of Altschul, et al.(1990) J Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389. Inanother embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the entirelength of the sequences aligned (i.e., a global alignment). A preferred,non-limiting example of a mathematical algorithm utilized for the globalcomparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM 120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or even 100% sequence identity, between the siRNA and theportion of the target mRNA is preferred. Alternatively, the siRNA may bedefined functionally as a nucleotide sequence (or oligonucleotidesequence) that is capable of hybridizing with a portion of the targetmRNA transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C. or 70° C. hybridization for 12-16 hours; followed by washing).Additional hybridization conditions include hybridization at 70° C. in1×SSC or 50° C. in 1×SSC, 50% formamide followed by washing at 70° C. in0.3×SSC or hybridization at 70° C. in 4×SSC or 50° C. in 4×SSC, 50%formamide followed by washing at 67° C. in 1×SSC. The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature (Tm) of thehybrid, where Tm is determined according to the following equations. Forhybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+Tbases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs inlength, Tm(° C.)=81.5+16.6 (log₁₀[Na+])+0.41 (% G+C)−(600/N), where N isthe number of bases in the hybrid, and [Na+] is the concentration ofsodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M).Additional examples of stringency conditions for polynucleotidehybridization are provided in Sambrook, J., E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11,and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al.,eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporatedherein by reference. The length of the identical nucleotide sequencesmay be at least about or about equal to 10, 12, 15, 17, 20, 22, 25, 27,30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.

In one embodiment, the siRNA molecules of the present invention aremodified to improve stability in serum or in growth medium for cellcultures. In order to enhance the stability, the 3′-residues may bestabilized against degradation, e.g., they may be selected such thatthey consist of purine nucleotides, particularly adenosine or guanosinenucleotides. Alternatively, substitution of pyrimidine nucleotides bymodified analogues, e.g., substitution of uridine by 2′-deoxythymidineis tolerated and does not affect the efficiency of RNA interference. Forexample, the absence of a 2′hydroxyl may significantly enhance thenuclease resistance of the siRNAs in tissue culture medium.

In another embodiment of the present invention the siRNA molecule maycontain at least one modified nucleotide analogue. The nucleotideanalogues may be located at positions where the target-specificactivity, e.g., the RNAi mediating activity is not substantiallyeffected, e.g., in a region at the 5′-end and/or the 3′-end of the RNAmolecule. Particularly, the ends may be stabilized by incorporatingmodified nucleotide analogues.

Nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In preferred backbone-modified ribonucleotides the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In preferred sugar modifiedribonucleotides, the 2′OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or NO₂, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

Nucleotide analogues also include nucleobase-modified ribonucleotides,i.e., ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

RNA may be produced enzymatically or by partial/total organic synthesis,any modified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, an siRNA is prepared chemically.Methods of synthesizing RNA molecules are known in the art, inparticular, the chemical synthesis methods as described in Verina andEckstein (1998), Annul Rev. Biochem. 67:99. In another embodiment, ansiRNA is prepared enzymatically. For example, an siRNA can be preparedby enzymatic processing of a long, double-stranded RNA having sufficientcomplementarity to the desired target mRNA. Processing of long RNA canbe accomplished in vitro, for example, using appropriate cellularlysates and siRNAs can be subsequently purified by gel electrophoresisor gel filtration. siRNA can then be denatured according toart-recognized methodologies. In an exemplary embodiment, siRNA can bepurified from a mixture by extraction with a solvent or resin,precipitation, electrophoresis, chromatography, or a combinationthereof. Alternatively, the siRNA may be used with no or a minimum ofpurification to avoid losses due to sample processing.

Alternatively, the siRNAs can also be prepared by enzymatictranscription from synthetic DNA templates or from DNA plasmids isolatedfrom recombinant bacteria. Typically, phage RNA polymerases are usedsuch as T7, T3 or SP6 RNA polyimerase (Milligan and Uhlenbeck (1989)Methods EnzynioL 180:51-62). The RNA may be dried for storage ordissolved in an aqueous solution. The solution may contain buffers orsalts to inhibit annealing, and/or promote stabilization of the doublestrands.

Commercially available design tools and kits, such as those availablefrom Ambion, Inc. (Austin, Tex.), and the Whitehead Institute ofBiomedical Research at MIT (Cambridge, Mass.) allow for the design andproduction of siRNA. By way of example, a desired mRNA sequence can beentered into a sequence program that will generate sense and antisensetarget strand sequences. These sequences can then be entered into aprogram that determines the sense and antisense siRNA oligonucleotidetemplates. The programs can also be used to add, e.g., hairpin insertsor Ti promoter primer sequences. Kits also can then be employed to buildsiRNA expression cassettes.

In various embodiments, siRNAs are synthesized in vivo, in situ, and invitro. Endogenous RNA polymerase of the cell may mediate transcriptionin vivo or in situ, or cloned RNA polymerase can be used fortranscription in vivo or in vitro. For transcription from a transgene invivo or an expression construct, a regulatory region (e.g., promoter,enhancer, silencer, splice donor and acceptor, polyadenylation) may beused to transcribe the siRNAs. Inhibition may be targeted by specifictranscription in an organ, tissue, or cell type; stimulation of anenvironmental condition (e.g., infection, stress, temperature, chemicalinducers); and/or engineering transcription at a developmental stage orage. A transgenic organism that expresses siRNAs from a recombinantconstruct may be produced by introducing the construct into a zygote, anembryonic stem cell, or another multipotent cell derived from theappropriate organism.

In one embodiment, the target mRNA of the invention specifies the aminoacid sequence of at least one protein such as a cellular protein (e.g.,a nuclear, cytoplasmic, transmembrane, or membrane-associated protein).In another embodiment, the target mRNA of the invention specifies theamino acid sequence of an extracellular protein (e.g., an extracellularmatrix protein or secreted protein). As used herein, the phrase“specifies the amino acid sequence” of a protein means that the mRNAsequence is translated into the amino acid sequence according to therules of the genetic code. The following classes of proteins are listedfor illustrative purposes: developmental proteins (e.g., adhesionmolecules, cyclin kinase inhibitors, Wnt family members, Pax familymembers, Winged helix family members, Hox family members,cytokines/lymphokines and their receptors, growth/differentiationfactors and their receptors, neurotransmitters and their receptors);oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2. CBL,CSFIR, ERBA, ERBB, EBRB2, ERBB2, ERBB3, ETSI, ETSI, ETV6, FGR, FOS, FYN,HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS,PIM 1, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor proteins(e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF 1, NF2, RB 1, TP53, and WTI);and enzymes (e.g., ACC synthases and oxidases, ACP desaturases andhydroxylases, ADPglucose pyrophorylases, acetylases and deacetylases,ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases,cellulases, chalcone synthases, chitinases, cyclooxygenases,decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases,glucanases, glucose oxidases, granule-bound starch synthases, GTPases,helicases, hemicellulases, integrases, inulinases, invertases,isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes,nopaline synthases, octopine synthases, pectinesterases, peroxidases,phosphatases, phospholipases, phosphorylases, phytases, plant growthregulator synthases, polygalacturonases, proteinases and peptidases,pullanases, recombinases, reverse transcriptases, RUBISCOs,topoisomerases, and xylanases), proteins involved in tumor growth(including vascularization) or in metastatic activity or potential,including cell surface receptors and ligands as well as secretedproteins, cell cycle regulatory, gene regulatory, and apoptosisregulatory proteins, immune response, inflammation, complement, orclotting regulatory proteins.

As used herein, the term “oncogene” refers to a gene which stimulatescell growth and, when its level of expression in the cell is reduced,the rate of cell growth is reduced or the cell becomes quiescent. In thecontext of the present invention, oncogenes include intracellularproteins, as well as extracellular growth factors which may stimulatecell proliferation through autocrine or paracrine function. Examples ofhuman oncogenes against which siRNA and morpholino constructs candesigned include c-myc, c-myb, mdm2, PKA-I (protein kinase A type I),Abl-1, Bcl2, Ras, c-Raf kinase, CDC25 phosphatases, cyclins, cyclindependent kinases (cdks), telomerase, PDGF/sis, erb-B, fos, jun, mos,and src, to name but a few. In the context of the present invention,oncogenes also include a fusion gene resulted from chromosomaltranslocation, for example, the Bcr/Abl fusion oncogene.

Further proteins include cyclin dependent kinases, c-myb, c-myc,proliferating cell nuclear antigen (PCNA), transforming growthfactor-beta (TGF-beta), and transcription factors nuclear factor kappaB(NF-.kappa.B), E2F, HER-2/neu, PKA, TGF-alpha, EGFR, TGF-beta, IGFIR,P12, MDM2, BRCA, Bcl-2, VEGF, MDR, ferritin, transferrin receptor, IRE,C-fos, HSP27, C-raf and metallothionein genes.

The siRNA employed in the present invention can be directed against thesynthesis of one or more proteins. Additionally or alternatively, therecan be more than one siRNA directed against a protein, e.g., duplicatesiRNA or siRNA that correspond to overlapping or non-overlapping targetsequences against the same target protein. Accordingly, in oneembodiment two, three, four or any plurality of siRNAs against the sametarget mRNA can be included in the nanoparticles of the invention.Additionally, several siRNAs directed against several proteins can beemployed. Alternatively, the siRNA can be directed against structural orregulatory RNA molecules that do not code for proteins.

In a preferred aspect of the invention, the target mRNA molecule of theinvention specifies the amino acid sequence of a protein associated witha pathological condition. For example, the protein may be apathogen-associated protein (e.g., a viral protein involved inimmunosuppression or immunoavoidance of the host, replication of thepathogen, transmission of the pathogen, or maintenance of theinfection), or a host protein which facilitates entry of the pathogeninto the host, drug metabolism by the pathogen or host, replication orintegration of the pathogen's genome, establishment or spread ofinfection in the host, or assembly of the next generation of pathogen.Alternatively, the protein may be a tumor-associated protein or anautoimmune disease-associated protein.

In one embodiment, the target mRNA molecule of the invention specifiesthe amino acid sequence of an endogenous protein (i.e. a protein presentin the genome of a cell or organism). In another embodiment, the targetmRNA molecule of the invention specifies the amino acid sequence of aheterologous protein expressed in a recombinant cell or a geneticallyaltered organism. In another embodiment, the target mRNA molecule of theinvention specifies the amino acid sequence of a protein encoded by atransgene (i.e., a gene construct inserted at an ectopic site in thegenome of the cell). In yet another embodiment, the target mRNA moleculeof the invention specifies the amino acid sequence of a protein encodedby a pathogen genome which is capable of infecting a cell or an organismfrom which the cell is derived.

By inhibiting the expression of such proteins, valuable informationregarding the function of said proteins and therapeutic benefits whichmay be obtained from said inhibition may be obtained.

In one embodiment, the nanoparticles of this invention comprises one ormore siRNA molecules to silence a PDGF beta gene, Erb-B gene, Src gene,CRK gene, GRB2 gene, RAS gene, MEKK gene, INK gene, RAF gene, Erkl/2gene, PCNA(p21) gene, MYB gene, JIJN gene, FOS gene, BCL-2 gene, CyclinD gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene,beta-catenin gene, c-MET gene, PKC gene, Skp2 gene, kinesin spindleprotein gene, Bcr-Abl gene, Stat3 gene, cSrc gene, PKC gene, Bax gene,Bcl-2 gene, EGFR gene, VEGF gene, myc gene, NFκB gene, STAT3 gene,survivin gene, Her2/Neu gene, topoisomerase I gene, PLK1 gene, proteinkinase 3 gene, CD31 gene, IGF-1 gene, topoisomerase II alpha gene,mutations in the p73 gene, mutations in the p21 (WAF 1/CIP 1) gene,mutations in the p27(KIP1) gene, mutations in the PPM1D gene, mutationsin the RAS gene, mutations in the caveolin I gene, mutations in the MIBI gene, mutations in the MTAI gene, mutations in the M68 gene, mutationsin tumor suppressor genes, mutations in the p53 tumor suppressor gene,mutations in the p53 family member DN-p63, mutations in the pRb tumorsuppressor gene, mutations in the APC1 tumor suppressor gene, mutationsin the BRCA1 tumor suppressor gene, mutations in the PTEN tumorsuppressor gene, mLL fusiongene, BCRIABL fusion gene, TEL/AML1 fusiongene, EWS/FLI1 fusion gene, TLS/FUS1 fusion gene, PAX3/FKHR fusion gene,AML1/ETO fusion gene, alpha v-integrin gene, Fit-i receptor gene,tubulin gene, Human Papilloma Virus gene, a gene required for HumanPapilloma Virus replication, Human Immunodeficiency Virus gene, a generequired for Human Immunodeficiency Virus replication, Hepatitis A Virusgene, a gene required for Hepatitis A Virus replication, Hepatitis BVirus gene, a gene required for Hepatitis B Virus replication, HepatitisC Virus gene, a gene required for Hepatitis C Virus replication,Hepatitis D Virus gene, a gene required for Hepatitis D Virusreplication, Hepatitis E Virus gene, a gene required for Hepatitis BVirus replication, Hepatitis F Virus gene, a gene required for HepatitisF Virus replication, Hepatitis G Virus gene, a gene required forHepatitis G Virus replication, Hepatitis H Virus gene, a gene requiredfor Hepatitis H Virus replication, Respiratory Syncytial Virus gene, agene that is required for Respiratory Syncytial Virus replication,Herpes Simplex Virus gene, a gene that is required for Herpes SimplexVirus replication, herpes Cytomegalovirus gene, a gene that is requiredfor herpes Cytomegalovirus replication, herpes Epstein Barr Virus gene,a gene that is required for herpes Epstein Barr Virus replication,Kaposi's Sarcoma-associated Herpes Virus gene, a gene that is requiredfor Kaposi's Sarcoma-associated Herpes Virus replication, JC Virus gene,human gene that is required for JC Virus replication, myxovirus gene, agene that is required for myxovirus gene replication, rhinovirus gene, agene that is required for rhinovirus replication, coronavirus gene, agene that is required for coronavirus replication, West Nile Virus gene,a gene that is required for West Nile Virus replication, St. LouisEncephalitis gene, a gene that is required for St. Louis Encephalitisreplication, Tick-borne encephalitis virus gene, a gene that is requiredfor Tick-borne encephalitis virus replication, Murray Valleyencephalitis virus gene, a gene that is required for Murray Valleyencephalitis virus replication, dengue virus gene, a gene that isrequired for dengue virus gene replication, Simian Virus 40 gene, a genethat is required for Simian Virus 40 replication, Human T CellLymphotropic Virus gene, a gene that is required for Human T CellLymphotropic Virus replication, Moloney-Murine Leukemia Virus gene, agene that is required for Moloney-Murine Leukemia Virus replication,encephalomyocarditis virus gene, a gene that is required forencephalomyocarditis virus replication, measles virus gene, a gene thatis required for measles virus replication, Vericella zoster virus gene,a gene that is required for Vericella zoster virus replication,adenovirus gene, a gene that is required for adenovirus replication,yellow fever virus gene, a gene that is required for yellow fever virusreplication, poliovirus gene, a gene that is required for poliovirusreplication, poxvirus gene, a gene that is required for poxvirusreplication, plasmodium gene, a gene that is required for plasmodiumgene replication, Mycobacterium ulcerans gene, a gene that is requiredfor Mycobacterium ulcerans replication, Mycobacterium tuberculosis gene,a gene that is required for Mycobacterium tuberculosis replication,Mycobacterium leprae gene,-185-a gene that is required for Mycobacteriumleprae replication, Staphylococcus aureus gene, a gene that is requiredfor Staphylococcus aureus replication, Streptococcus pneumoniae gene, agene that is required for Streptococcus pneumoniae replication,Streptococcus pyogenes gene, a gene that is required for Streptococcuspyogenes replication, Chlamydia pneumoniae gene, a gene that is requiredfor Chlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a genethat is required for Mycoplasma pneumoniae replication, an integringene, a selectin gene, complement system gene, chemokine gene, chemokinereceptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIGgene, Pro-Platelet Basic Protein gene, MIP-11 gene, MIP-1J gene, RANTESgene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,CMBKR3 gene, CMBKR5v, AIF-1 gene, 1-309 gene, a gene to a component ofan ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene,DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene,SCA8 gene, allele gene found in LOH cells, or one allele gene of apolymorphic gene. Examples of relevant siRNA molecules to silence genesand methods of making siRNA molecules can be found from commercialsources such as Dharmacon or from the following patent applications:US2005017667, WO2006066158, WO2006078278, U.S. Pat. No. 7,056,704, U.S.Pat. No. 7,078,196, U.S. Pat. No. 5,898,031, U.S. Pat. No. 6,107,094, EP1144623, and EU 1144623, all of which are incorporated by reference intheir entireties. While a number of specific gene silencing targets arelisted, this list is merely illustrative and other siRNA molecules couldalso be used with the nanoparticles of this invention.

In one embodiment, the nanoparticles of this invention comprise an siRNAmolecule having RNAi activity against an RNA, wherein the siRNA moleculecomprises a sequence complementary to any RNA having coding ornon-encoding sequence, such as those sequences referred to by GenBankAccession Nos. described in Table V of PCT/US03/05028 (International PCTPublication No. WO 03/4654) or otherwise known in the art.

In one embodiment, the nanoparticles of this invention comprise an siRNAmolecule which silences the vascular endothelial growth factor gene. Inanother embodiment, the nanoparticles of this invention comprise ansiRNA molecule which silences the vascular endothelial growth factorreceptor gene.

In another embodiment, the nanoparticles of this invention comprise ansiRNA molecule, wherein the sequence of the siRNA molecule iscomplementary to tumor-related targets, including, but not limited to,hypoxia-inducible factor-1 (HIF-1), which is found in human metastaticprostate PC3-M cancer cells (Mol. Carcinog. 2008 Jan. 31 [Epub ahead ofprint]); the HIF-1 downstream target gene (Mol. Carcinog. 2008 Jan. 31[Epub ahead of print]), mitogen-activated protein kinases (MAPKs),hepatocyte growth factor (HGF), interleukin 12p70 (IL12),glucocorticoid-induced tumor necrosis factor receptor (GITR),intercellular adhesion molecule 1 (ICAM-1), neurotrophin-3 (NT-3),interleukin 17 (IL17), interleukin 18 binding protein a (IL18 Bpa) andepithelial-neutrophil activating peptide (ENA78) (see, e.g., “Cytokineprofiling of prostatic fluid from cancerous prostate glands identifiescytokines associated with extent of tumor and inflammation”, TheProstate Early view Published Online: 24 Mar. 2008); PSMA (see, e.g.,“Cell-Surface labeling and internalization by a fluorescent inhibitor ofprostate-specific membrane antigen” The Prostate Early view PublishedOnline: 24 Mar. 2008); Androgen receptor (AR), keratin, epithelialmembrane antigen, EGF receptor, and E cadherin (see, e.g.,“Characterization of PacMetUT1, a recently isolated human prostatecancer cell line”); peroxisomes proliferators-activated receptor γ(PPARγ; see e.g., The Prostate Volume 68, Issue 6, Date: 1 May 2008,Pages: 588-598); the receptor for advanced glycation end products (RAGE)and the advanced glycation end products (AGE), (see, e.g., “V domain ofRAGE interacts with AGEs on prostate carcinoma cells” The Prostate Earlyview Published Online: 26 Feb. 2008); the receptor tyrosine kinaseerb-B2 (Her2/neu), hepatocyte growth factor receptor (Met), transforminggrowth factor-beta 1 receptor (TGFβR1), nuclear factor kappa B (NFκB),Jagged-1, Sonic hedgehog (Shh), Matrix metalloproteinases (MMPs, esp.MMP-7), Endothelin receptor type A (ET_(A)), Endothelin-1 (ET-1),Nuclear receptor subfamily 3, group C, member 1 (NR3C1), Nuclearreceptor co-activator 1 (NCOA1), NCOA2, NCOA3, E1A binding protein p300(EP300), CREB binding protein (CREBBP), Cyclin G associated kinase(GAK), Gelsolin(GSN), Aldo-keto reductase family 1, member C1 (AKR1C1),AKR1C2, AKR1C3, Neurotensin(NTS), Enolase 2(ENO2), Chromogranin B (CHGB,secretogranin 1), Secretagogin (SCGN, or EF-hand calcium bindingprotein), Dopa decarboxylase(DDC, or aromatic L-amino aciddecarboxylase), steroid receptor co-activator-1 (SRC-1), SRC-2 (a.k.a.TIF2), SRC-3 (a.k.a. AIB-1) (see, e.g., “Longitudinal analysis ofandrogen deprivation of prostate cancer cells identifies pathways toandrogen independence” The Prostate Early view Published Online: 26 Feb.2008); estrogen receptors (ERα, ERβ or GPR30) (see, e.g., The ProstateVolume 68, Issue 5, Pages 508-516); the melanoma cell adhesion molecule(MCAM) (see, e.g., The Prostate Volume 68, Issue 4, Pages 418-426;angiogenic factors (such as vascular endothelial growth factor (VEGF)and erythropoietin), glucose transporters (such as GLUT1),BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) (see, e.g., TheProstate Volume 68, Issue 3, Pages 336-343); types 1 and 2 5α-reductase(see, e.g., The Journal of Urology Volume 179, Issue 4, Pages1235-1242); ERG and ETV1, prostate-specific antigen (PSA),prostate-specific membrane antigen (PSMA), prostate stem cell antigen(PSCA), α-Methylacyl coenzyme A racemase (AMACR), PCA3^(DD3)glutathione-S-transferase, pi 1 (GSTP1), p16, ADP-ribosylation factor(ARF), O-6-methylguanine-DNA methyltransferase (MGMT), human telomerasereverse transcriptase (hTERT), early prostate cancer antigen (EPCA),human kallikrein 2 (HK2) and hepsin (see, e.g., The Journal of UrologyVolume 178, Issue 6, Pages 2252-2259); bromodomain containing 2 (BRD2),eukaryotic translation initiation factor 4 gamma, 1 (eIF4G1), ribosomalprotein L13a (RPL13a), and ribosomal protein L22 (RPL22) (see, e.g., NEngl J Med 353 (2005), p. 1224); HER2/neu, Derlin-1, ERBB2, AKT,cyclooxygenase-2 (COX-2), PSMD3, CRKRS, PERLD1, and C17ORF37, PPP4C,PARN, ATP6V0C, C16orf14, GBL, HAGH, ITFG3, MGC13114, MRPS34, NDUFB10,NMRAL1, NTHL1, NUBP2, POLR3K, RNPS1, STUB1, TBL3, and USP7. All of thereferences described herein are incorporated herein by reference intheir entireties.

Thus, in one embodiment, the invention comprises a nanoparticlecomprising a targeting moiety (e.g., CREKA, an aptamer, or affibody), abiodegradable polymer, a stealth polymer, and an siRNA molecule. In oneembodiment, the invention comprises a nanoparticle comprising atargeting moiety (e.g., CREKA, an aptamer, or affibody), a biodegradablepolymer, a stealth component, and an siRNA molecule that silences thevascular endothelial growth factor gene. In one embodiment, theinvention comprises a nanoparticle comprising a targeting moiety (e.g.,CREKA, an aptamer, or affibody), a biodegradable polymer, a stealthcomponent, and an siRNA molecule that silences the vascular endothelialgrowth factor receptor gene. In another embodiment, the inventioncomprises a nanoparticle comprising a targeting moiety (e.g., CREKA, anaptamer, or affibody), PLGA, polyethylene glycol, and an siRNA molecule.In one embodiment, the invention comprises a nanoparticle comprising atargeting moiety (e.g., CREKA, an aptamer, or affibody), a biodegradablepolymer, a stealth component, and an siRNA molecule wherein thenanoparticle can selectively accumulate in the prostate or in thevascular endothelial tissue surrounding a cancer. In one embodiment, theinvention comprises a nanoparticle comprising a targeting moiety (e.g.,CREKA, an aptamer, or affibody), a biodegradable polymer, a stealthcomponent, and an siRNA molecule wherein the nanoparticle canselectively accumulate in the prostate or in the vascular endothelialtissue surrounding a cancer and wherein the nanoparticle can beendocytosed by a PSMA expressing cell.

In another embodiment, the siRNA that is incorporated into thenanoparticle of the invention are those that treat prostate cancer, suchas those disclosed in U.S. application Ser. No. 11/021,159 (siRNAsequence is complementary to SEQ ID No.8: gaaggccagu uguauggac), andU.S. application Ser. No. 11/349,473 (discloses siRNAs that bind to aregion from nucleotide 3023 to 3727 of SEQ ID No. 1). Both of thesereferences are incorporated herein by reference in their entirety.

In another embodiment, the therapeutic agents of the nanoparticles ofthe invention include RNAs that can be used to treat cancer, such asanti-sense mRNAs and microRNAs. Examples of microRNAs that can be usedas therapeutic agents for the treatment of cancer include thosedisclosed in Nature 435 (7043): 828-833; Nature 435 (7043): 839-843; andNature 435 (7043): 834-838, all of which are incorporated herein byreference in their entireties.

In one embodiment, the invention specifically excludes nanoparticlescontaining iron oxide. In one embodiment, blood clotting does not occurat the location where the nanoparticle accumulates.

In one embodiment, the therapeutic agents used in conjunction with thenanoparticles of the invention include one or more agents useful for thetreatment of restenosis. Examples of such agents include, but are notlimited to, everolimus, paclitaxel, zotarolimus, pioglitazone, BO-653,rosiglitazone, sirolimus, dexamethasone, rapamycin, tacrolimus,biophosphonates, estrogen, angiopeptin, statin, PDGF inhibitors, ROCKinhibitors, MMP inhibitors, and 2-CdA. In a certain embodiment, thetherapeutic agents useful for the treatment of restenosis arezotarolimus and dexamethasone, including combinations of zotarolimus anddexamethasone.

In a preferred embodiment, the nanoparticles of the invention can bedelivered to or near a vulnerable plaque using a medical device such asa needle catheter, drug eluding stent or stent graft. Such devices arewell known in the art, and are described, for example, in U.S. Pat. No.7,008,411, which is incorporated herein by reference in its entirety. Inone embodiment, a drug eluting stent and/or needle catheter may beimplanted at the region of vessel occlusion that may be upstream from avulnerable plaque region. A medical device, such as a drug elutingstent, needle catheter, or stent graft may be used to treat theocclusive atherosclerosis (i.e., non-vulnerable plaque) while releasingthe nanoparticle of the invention to treat a vulnerable plaque regiondistal or downstream to the occlusive plaque. The nanoparticle may bereleased slowly over time.

The nanoparticles of the invention can also be delivered to a subject inneed thereof using the Genie™ balloon catheter available from Acrostak(http://www.acrostak.com/genie_en.htm). The nanoparticles of theinvention can also be delivered to a subject in need thereof usingdelivery devices that have been developed for endovascular local genetransfer such as passive diffusion devices (e.g., double-occlusionballoon, spiral balloon), pressure-driven diffusion devices (e.g.,microporous balloon, balloon-in-balloon devices, double-layer channeledperfusion balloon devices, infusion-sleeve catheters, hydrogel-coatedballoons), and mechanically or electrically enhanced devices (e.g.,needle injection catheter, iontophoretic electric current-enhancedballoons, stent-based system), or any other delivery system disclosed inRadiology 2003; 228:36-49, or Int J Nanomedicine 2007; 2(2):143-61,which are incorporated herein by reference in their entirety.

Once the inventive conjugates have been prepared, they may be combinedwith pharmaceutical acceptable carriers to form a pharmaceuticalcomposition, according to another aspect of the invention. As would beappreciated by one of skill in this art, the carriers may be chosenbased on the route of administration as described below, the location ofthe target issue, the drug being delivered, the time course of deliveryof the drug, etc.

Methods of Treatment

In some embodiments, targeted particles in accordance with the presentinvention may be used to treat, alleviate, ameliorate, relieve, delayonset of, inhibit progression of, reduce severity of, and/or reduceincidence of one or more symptoms or features of a disease, disorder,and/or condition. In some embodiments, inventive targeted particles maybe used to treat cancer, e.g., breast cancer, and/or cancer cells, e.g.,breast cancer cells.

The term “cancer” includes pre-malignant as well as malignant cancers.Cancers include, but are not limited to, prostate, gastric cancer,colorectal cancer, skin cancer, e.g., melanomas or basal cellcarcinomas, lung cancer, cancers of the head and neck, bronchus cancer,pancreatic cancer, urinary bladder cancer, brain or central nervoussystem cancer, peripheral nervous system cancer, esophageal cancer,cancer of the oral cavity or pharynx, liver cancer, kidney cancer,testicular cancer, biliary tract cancer, small bowel or appendix cancer,salivary gland cancer, thyroid gland cancer, adrenal gland cancer,osteosarcoma, chondrosarcoma, cancer of hematological tissues, and thelike. “Cancer cells” can be in the form of a tumor, exist alone within asubject (e.g., leukemia cells), or be cell lines derived from a cancer.

Cancer can be associated with a variety of physical symptoms. Symptomsof cancer generally depend on the type and location of the tumor. Forexample, lung cancer can cause coughing, shortness of breath, and chestpain, while colon cancer often causes diarrhea, constipation, and bloodin the stool. However, to give but a few examples, the followingsymptoms are often generally associated with many cancers: fever,chills, night sweats, cough, dyspnea, weight loss, loss of appetite,anorexia, nausea, vomiting, diarrhea, anemia, jaundice, hepatomegaly,hemoptysis, fatigue, malaise, cognitive dysfunction, depression,hormonal disturbances, neutropenia, pain, non-healing sores, enlargedlymph nodes, peripheral neuropathy, and sexual dysfunction.

In one aspect of the invention, a method for the treatment of cancer(e.g. breast cancer) is provided. In some embodiments, the treatment ofcancer comprises administering a therapeutically effective amount ofinventive targeted particles to a subject in need thereof, in suchamounts and for such time as is necessary to achieve the desired result.In certain embodiments of the present invention a “therapeuticallyeffective amount” of an inventive targeted particle is that amounteffective for treating, alleviating, ameliorating, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of cancer.

In one aspect of the invention, a method for administering inventivecompositions to a subject suffering from cancer (e.g. breast cancer) isprovided. In some embodiments, particles to a subject in such amountsand for such time as is necessary to achieve the desired result (i.e.,treatment of cancer). In certain embodiments of the present invention a“therapeutically effective amount” of an inventive targeted particle isthat amount effective for treating, alleviating, ameliorating,relieving, delaying onset of, inhibiting progression of, reducingseverity of, and/or reducing incidence of one or more symptoms orfeatures of cancer.

In other embodiments, the nanoparticles of the present invention can beused to inhibit the growth of cancer cells, e.g., breast cancer cells.As used herein, the term “inhibits growth of cancer cells” or“inhibiting growth of cancer cells” refers to any slowing of the rate ofcancer cell proliferation and/or migration, arrest of cancer cellproliferation and/or migration, or killing of cancer cells, such thatthe rate of cancer cell growth is reduced in comparison with theobserved or predicted rate of growth of an untreated control cancercell. The term “inhibits growth” can also refer to a reduction in sizeor disappearance of a cancer cell or tumor, as well as to a reduction inits metastatic potential. Preferably, such an inhibition at the cellularlevel may reduce the size, deter the growth, reduce the aggressiveness,or prevent or inhibit metastasis of a cancer in a patient. Those skilledin the art can readily determine, by any of a variety of suitableindicia, whether cancer cell growth is inhibited.

Inhibition of cancer cell growth may be evidenced, for example, byarrest of cancer cells in a particular phase of the cell cycle, e.g.,arrest at the G2/M phase of the cell cycle. Inhibition of cancer cellgrowth can also be evidenced by direct or indirect measurement of cancercell or tumor size. In human cancer patients, such measurementsgenerally are made using well known imaging methods such as magneticresonance imaging, computerized axial tomography and X-rays. Cancer cellgrowth can also be determined indirectly, such as by determining thelevels of circulating carcinoembryonic antigen, prostate specificantigen or other cancer-specific antigens that are correlated withcancer cell growth. Inhibition of cancer growth is also generallycorrelated with prolonged survival and/or increased health andwell-being of the subject.

The present invention is directed, in part, to the discovery that acollagen IV alpha-2 chain related polypeptide can act as a receptor forthe CREKA tumor targeting peptide. Collagens are a major component ofthe extracellular matrix (ECM), an interconnected molecular networkproviding mechanical support for cells and tissues and regulatingbiochemical and cellular processes such as adhesion, migration, geneexpression and differentiation (see, e.g., U.S. Patent Application No.2005/0048063, which is incorporated herein by reference in itsentirety). In higher animals, at least 19 distinct collagen typesdiffering in their higher order structures and functions have beenidentified based on the presence of the characteristic collagentriple-helix structure. The collagens are sometimes categorized into thefibrillar and nonfibrillar collagens. The fibrillar (interstitial)collagens include types I, II, III, V and XI, while the nonfibrillarcollagens include types IV, VI, IX, X, XI, XII, XIV and XIII.

As a non-limiting example, a method of the invention for treating cancercan be useful for treating breast cancer. Targeting poly(amio acids)useful in the invention include those which selectively target tumorvasculature and selectively bind non-helical collagen. Targetingpoly(amio acids) useful in the invention also include those whichselectively target to tumor vasculature and selectively bind collagenIV, and those which selectively target tumor vasculature and selectivelybind denatured collagen IV in preference to native collagen IV.

Inventive therapeutic protocols involve administering a therapeuticallyeffective amount of an inventive targeted particle to a healthyindividual (i.e., a subject who does not display any symptoms of cancerand/or who has not been diagnosed with cancer). For example, healthyindividuals may be “immunized” with an inventive targeted particle priorto development of cancer and/or onset of symptoms of cancer; at riskindividuals (e.g., patients who have a family history of cancer;patients carrying one or more genetic mutations associated withdevelopment of cancer; patients having a genetic polymorphism associatedwith development of cancer; patients infected by a virus associated withdevelopment of cancer; patients with habits and/or lifestyles associatedwith development of cancer; etc.) can be treated substantiallycontemporaneously with (e.g., within 48 hours, within 24 hours, orwithin 12 hours of) the onset of symptoms of cancer. Of courseindividuals known to have cancer may receive inventive treatment at anytime.

In another aspect, the invention provides a method of treatingcardiovascular conditions in a subject in need thereof, comprisingadministering to the subject an effective amount of thecontrolled-release system of the invention. Such cardiovascularconditions include, but are not limited to, restenosis and vulnerableplaque. In one embodiment, the nanoparticles of this invention aredelivered locally to the coronary arteries, central arteries, peripheralarteries, veins, and bile ducts. In another embodiment, thenanoparticles of this invention are delivered locally to the coronaryarteries, central arteries, peripheral arteries, veins, and bile ductsafter the implantation of a stent in such tissue in a patient for thetreatment of restenosis. In another embodiment, the nanoparticles ofthis invention are administered to a patient undergoing a coronaryangioplasty, a peripheral angioplasty, a renal artery angioplasty, or acarotid angioplasty in order to prevent resenosis.

In one embodiment, the nanoparticles of this invention pass through theendothelial layer of a blood vessel due to plaque damage of theendothelial tissue and bind to the basement membrane.

In another aspect, the invention provides a method of treatingrestenosis in a subject in need thereof, comprising administering to thesubject an effective amount of the controlled-release system of theinvention. In one embodiment, the controlled-release system is locallyadministered to a designated region of the blood vessel where therestenosis occurs. In still another embodiment, the controlled-releasesystem is administered via a medical device. In yet another embodiment,the medical device is a drug eluding stent, needle catheter, or stentgraft. In another embodiment, the invention provides a method oftreating restenosis in a subject in need thereof, comprisingadministering to the subject an effective amount of thecontrolled-release system of the invention wherein the controlledrelease system contains a drug selected from the group consisting ofeverolimus, paclitaxel, zotarolimus, pioglitazone, BO-653,rosiglitazone, sirolimus, dexamethasone, rapamycin, tacrolimus,biophosphonates, estrogen, angiopeptin, statin, PDGF inhibitors, ROCKinhibitors, MMP inhibitors, and 2-CdA. In another embodiment, theinvention provides a method of treating restenosis in a subject in needthereof, comprising administering to the subject an effective amount ofthe controlled-release system of the invention wherein the controlledrelease system contains two drugs selected from everolimus, paclitaxel,zotarolimus, pioglitazone, BO-653, rosiglitazone, sirolimus,dexamethasone, rapamycin, tacrolimus, biophosphonates, estrogen,angiopeptin, statin, PDGF inhibitors, ROCK inhibitors, MMP inhibitors,and 2-CdA. In another embodiment, the invention provides a method oftreating restenosis in a subject in need thereof, comprisingadministering to the subject an effective amount of thecontrolled-release system of the invention wherein the controlledrelease system contains zotarolimus and dexamethasone.

In one embodiment, the nanoparticles of this invention are deliveredlocally to the coronary arteries, central arteries, peripheral arteries,veins, and bile ducts. In another embodiment, the nanoparticles of thisinvention are delivered locally to the coronary arteries, centralarteries, peripheral arteries, veins, and bile ducts after theimplantation of a stent in such tissue in a patient for the treatment ofrestenosis. In another embodiment, the nanoparticles of this inventionare administered to a patient undergoing a coronary angioplasty, aperipheral angioplasty, a renal artery angioplasty, or a carotidangioplasty in order to prevent resenosis. In another embodiment, thenanoparticles of this invention are administered within 12 hours of apatient undergoing a coronary angioplasty, a peripheral angioplasty, arenal artery angioplasty, or a carotid angioplasty in order to preventresenosis. In another embodiment, the nanoparticles of this inventionare administered locally to a patient undergoing a coronary angioplasty,a peripheral angioplasty, a renal artery angioplasty, or a carotidangioplasty in order to prevent resenosis.

Pharmaceutical Compositions

As used herein, the term “pharmaceutically acceptable carrier” means anon-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Remington'sPharmaceutical Sciences. Ed. by Gennaro, Mack Publishing, Easton, Pa.,1995 discloses various carriers used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Someexamples of materials which can serve as pharmaceutically acceptablecarriers include, but are not limited to, sugars such as lactose,glucose, and sucrose; starches such as com starch and potato starch;cellulose and its derivatives such as sodium carboxymethyl cellulose,ethyl cellulose, and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients such as cocoa butter and suppository waxes;oils such as peanut oil, cottonseed oil; safflower oil; sesame oil;olive oil; corn oil and soybean oil; glycols such as propylene glycol;esters such as ethyl oleate and ethyl laurate; agar; detergents such asTWEEN™ 80; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. If filtration or otherterminal sterilization methods are not feasible, the formulations can bemanufactured under aseptic conditions.

The pharmaceutical compositions of this invention can be administered toa patient by any means known in the art including oral and parenteralroutes. In certain embodiments parenteral routes are desirable sincethey avoid contact with the digestive enzymes that are found in thealimentary canal. According to such embodiments, inventive compositionsmay be administered by injection (e.g., intravenous, subcutaneous orintramuscular, intraperitoneal injection), rectally, vaginally,topically (as by powders, creams, ointments, or drops), or by inhalation(as by sprays).

In one embodiment, the nanoparticles of the present invention areadministered to a subject in need thereof systemically, e.g., by IVinfusion or injection. In a particular embodiment, the nanoparticles ofthe present invention are locally administered to a subject in needthereof. As used herein, “local administration” is when nanoparticles ofthe invention are brought into contact with the blood vessel wall orvascular tissue through a device (e.g., a stent).

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Inone embodiment, the inventive conjugate is suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) TWEEN™80. The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration may be suppositorieswhich can be prepared by mixing the inventive conjugate with suitablenon-irritating excipients or carriers such as cocoa butter, polyethyleneglycol, or a suppository wax which are solid at ambient temperature butliquid at body temperature and therefore melt in the rectum or vaginalcavity and release the inventive conjugate.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The inventiveconjugate is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives or buffers as may berequired. Ophthalmic formulations, ear drops, and eye drops are alsocontemplated as being within the scope of this invention. The ointments,pastes, creams, and gels may contain, in addition to the inventiveconjugates of this invention, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof. Transdermal patches have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms can be made by dissolving or dispensing the inventiveconjugates in a proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the inventive conjugates in a polymer matrix or gel.

Powders and sprays can contain, in addition to the inventive conjugatesof this invention, excipients such as lactose, talc, silicic acid,aluminum hydroxide, calcium silicates, and polyamide powder, or mixturesthereof. Sprays can additionally contain customary propellants such aschlorofluorohydrocarbons.

When administered orally, the inventive nanoparticles can be, but arenot necessarily, encapsulated. A variety of suitable encapsulationsystems are known in the art (“Microcapsules and Nanoparticles inMedicine and Pharmacy,” Edited by Doubrow, M., CRC Press, Boca Raton,1992; Mathiowitz and Langer J. Control. Release 5:13, 1987; Mathiowitzet al. Reactive Polymers 6:275, 1987; Mathiowitz et al. J. Appl. PolymerSci. 35:755, 1988; Langer Ace. Chem. Res. 33:94, 2000; Langer J.Control. Release 62:7, 1999; Uhrich et al. Chem. Rev. 99:3181, 1999;Zhou et al. J. Control. Release 75:27, 2001; and Hanes et al. Pharm.Biotechnol. 6:389, 1995). The inventive conjugates may be encapsulatedwithin biodegradable polymeric microspheres or liposomes. Examples ofnatural and synthetic polymers useful in the preparation ofbiodegradable microspheres include carbohydrates such as alginate,cellulose, polyhydroxyalkanoates, polyamides, polyphosphazenes,polypropylfumarates, polyethers, polyacetals, polycyanoacry lates,biodegradable polyurethanes, polycarbonates, polyanhydrides,polyhydroxyacids, poly(ortho esters), and other biodegradablepolyesters. Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides.

Pharmaceutical compositions for oral administration can be liquid orsolid. Liquid dosage forms suitable for oral administration of inventivecompositions include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and elixirs. In additionto an encapsulated or unencapsulated conjugate, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants, wetting agents, emulsifying andsuspending agents, sweetening, flavoring, and perfuming agents. As usedherein, the term “adjuvant” refers to any compound which is anonspecific modulator of the immune response. In certain embodiments,the adjuvant stimulates the immune response. Any adjuvant may be used inaccordance with the present invention. A large number of adjuvantcompounds is known in the art (Allison Dev. Biol. Stand. 92:3-11, 1998;Unkeless et al. Annu. Rev. Immunol. 6:251-281, 1998; and Phillips et al.Vaccine 10:151-158, 1992).

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, theencapsulated or unencapsulated conjugate is mixed with at least oneinert, pharmaceutically acceptable excipient or carrier such as sodiumcitrate or dicalcium phosphate and/or (a) fillers or extenders such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid, (b)binders such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectantssuch as glycerol, (d) disintegrating agents such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate, (e) solution retarding agents such as paraffin,(f) absorption accelerators such as quaternary ammonium compounds, (g)wetting agents such as, for example, cetyl alcohol and glycerolmonostearate, (h) absorbents such as kaolin and bentonite clay, and (i)lubricants such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof. Inthe case of capsules, tablets, and pills, the dosage form may alsocomprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart.

It will be appreciated that the exact dosage of the targeted particle ischosen by the individual physician in view of the patient to be treated,in general, dosage and administration are adjusted to provide aneffective amount of the targeted particle to the patient being treated.As used herein, the “effective amount” of an targeted particle refers tothe amount necessary to elicit the desired biological response. As willbe appreciated by those of ordinary skill in this art, the effectiveamount of targeted particle may vary depending on such factors as thedesired biological endpoint, the drug to be delivered, the targettissue, the route of administration, etc. For example, the effectiveamount of targeted particle containing an anti-cancer drug might be theamount that results in a reduction in tumor size by a desired amountover a desired period of time. Additional factors which may be takeninto account include the severity of the disease state; age, weight andgender of the patient being treated; diet, time and frequency ofadministration; drug combinations; reaction sensitivities; andtolerance/response to therapy.

The nanoparticles of the invention may be formulated in dosage unit formfor ease of administration and uniformity of dosage. The expression“dosage unit form” as used herein refers to a physically discrete unitof nanoparticle appropriate for the patient to be treated. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. For any nanoparticle, thetherapeutically effective dose can be estimated initially either in cellculture assays or in animal models, usually mice, rabbits, dogs, orpigs. The animal model is also used to achieve a desirable concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic efficacy and toxicity of nanoparticles can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose is therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose is lethal to 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceutical compositionswhich exhibit large therapeutic indices may be useful in someembodiments. The data obtained from cell culture assays and animalstudies can be used in formulating a range of dosage for human use.

The present invention also provides any of the above-mentionedcompositions in kits, optionally with instructions for administering anyof the compositions described herein by any suitable technique aspreviously described, for example, orally, intravenously, pump orimplantable delivery device, or via another known route of drugdelivery. “Instructions” can define a component of promotion, andtypically involve written instructions on or associated with packagingof compositions of the invention. Instructions also can include any oralor electronic instructions provided in any manner.

The “kit” typically defines a package including any one or a combinationof the compositions of the invention and the instructions, but can alsoinclude the composition of the invention and instructions of any formthat are provided in connection with the composition in a manner suchthat a clinical professional will clearly recognize that theinstructions are to be associated with the specific composition.

The kits described herein may also contain one or more containers, whichmay contain the inventive composition and other ingredients aspreviously described. The kits also may contain instructions for mixing,diluting, and/or administrating the compositions of the invention insome cases. The kits also can include other containers with one or moresolvents, surfactants, preservative and/or diluents (e.g., normal saline(0.9% NaCl), or 5% dextrose) as well as containers for mixing, dilutingor administering the components in a sample or to a subject in need ofsuch treatment.

The compositions of the kit may be provided as any suitable form, forexample, as liquid solutions or as dried powders. When the compositionprovided is a dry powder, the composition may be reconstituted by theaddition of a suitable solvent, which may also be provided. Inembodiments where liquid forms of the composition are used, the liquidform may be concentrated or ready to use. The solvent will depend on thenanoparticle and the mode of use or administration. Suitable solventsfor drug compositions are well known, for example as previouslydescribed, and are available in the literature. The solvent will dependon the nanoparticle and the mode of use or administration.

The invention also involves, in another aspect, promotion of theadministration of any of the nanoparticle described herein. In someembodiments, one or more compositions of the invention are promoted forthe prevention or treatment of various diseases such as those describedherein via administration of any one of the compositions of the presentinvention. As used herein, “promoted” includes all methods of doingbusiness including methods of education, hospital and other clinicalinstruction, pharmaceutical industry activity including pharmaceuticalsales, and any advertising or other promotional activity includingwritten, oral and electronic communication of any form, associated withcompositions of the invention.

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

EXAMPLES

The invention is further illustrated by the following examples. Theexamples should not be construed as further limiting.

Example 1 Amphiphilic Nanoparticle with Aptamer

In one embodiment, the A10 RNA aptamer which binds to the ProstateSpecific Membrane Antigen (PSMA) on the surface of prostate cancer cellsis conjugated to DSPE(1,2-Distearoyl-sn-glycero-3-phosphoethanolamine)-PEG-COOH using EDC/NHSchemistry with a conjugate concentration of 0.7 mg/mL. 0.21 mg of thisDSPE-PEG-aptamer bioconjugate is mixed with 0.07 mg lecithin in 2 mLaqueous solution containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolicacid) (PLGA, Mw=100 kD) is dissolved in 1 mL tetrahydrofuran (THF)solvent, to which 5% docetaxel of the mass of PLGA is added. This PLGAsolution is then mixed with the aqueous solution oflecithin/DSPE-PEG-Aptamer. These mixtures are vortexed for 3 minutes,followed by stirring for 2 hours. In order to remove all organicsolvents, these mixtures are then dialyzed for another 4 hours againstPBS buffer. This procedure would yield nanoparticles targeting toprostate cancer cells expressing PSMA antigens.

In a second embodiment, poly(D,L-lactic-co-glycolic acid) (PLGA) is usedas a polymeric core, lecithin monolayer (˜2.5 nm) as a lipid shell,poly(ethylene glycol) (PEG) as a stealth material, and the A10 RNAaptamer were used to develop targeted PLGA-Lecithin-PEG nanoparticles(NPs). Particle size could be tuned within the range from 40 nm to 500nm, accompanied with a surface zeta potential ranging from −80 mV to −30mV. Using docetaxel (a widely used chemotherapeutics for cancers) as amodel small molecule hydrophobic drug, the PLGA-Lecithin-PEG NP had drugencapsulation efficiency around 65% as contrast to 19% for theconventional PLGA-b-PEG diblock copolymer NP. In addition, less than 20%drugs were released from the NP during the first 6 hours, which holdsbroad promise for clinical applications. Both in vitro and in vivoresults demonstrated that the attached RNA aptamer effectively targetedPLGA-Lecithin-PEG NPs to prostate cancer cells which express PSMAantigen on their plasma membrane, such as LNCaP cells. FIG. 4 shows aschematic illustration of amphiphilic compound assisted polymericnanoparticles for targeted drug delivery. FIGS. 5A and 5B show size andzeta-potential stabilities of nanoparticles prepared according to thisexample. FIGS. 6 and 7 demonstrate drug encapsulation efficiency of alipid assisted polymeric nanoparticle as compared with a non-lipidassisted polymeric nanoparticle. FIG. 8 shows a drug release profile fora nanoparticle prepared according to this example.

Encapsulation efficiency is determined by taking a known amount of DNA,encapsulating it into a nanoparticle, removing any unencapsulated DNA byfiltration, lysing the nanoparticle, then detecting the amount of DNAthat was encapsulated by measuring its absorbance of light at 260 nm.The encapsulation efficiency is calculated by taking the amount of DNAthat was encapsulated, then dividing it by the amount of DNA that webegan with. Stated alternatively, it is the fraction of initial DNA thatis successfully encapsulated.

Zeta potential is determined by Quasi-elastic laser light scatteringwith a ZetaPALS dynamic light scattering detector (BrookhavenInstruments Corporation, Holtsville, N.Y.; 15 mW laser, incidentbeam=676 nm).

Example 2 Amphiphilic Nanoparticle with CREKA

The peptide CREKA is conjugated to DSPE-PEG-Maleimide before formulatingnanoparticles using the protocol of Example 1. This peptide will targetthe delivery and uptake of the nanoparticles to extracellular basementmembranes which are exposed under the leaky endothelial layer coveringatherosclerotic plaques.

Example 3 Amphiphilic Nanoparticle with AXYLZZLN

The peptide AXYLZZLN, or conservative variants or peptidomimeticsthereof, wherein X and Z are variable amino acids, can be conjugated toDSPE-PEG-Maleimide before formulating nanoparticles using the protocolof Example 1. This peptide will target the delivery and uptake of thenanoparticles to extracellular basement membranes which are exposedunder the leaky endothelial layer covering atherosclerotic plaques.

Example 4 Anti-Her2/AKERC-Targeted Nanoparticle Triblock PolymerSynthesis

Maleimide terminal poly(D,L-lactide)-block-poly(ethylene glycol)(PLA-PEG-MAL) (Mw˜10 kDa determined by GPC) was synthesized by ringopening polymerization. Carboxylic acid terminal poly(D,L-lactide)and/or poly(lactic-co-glycolic acid) was purchased from the DURECTcorporation (Pelham, Ala.). Bifunctional PEG (HO-PEG-MAL) was purchasedfrom Nektar Therapeutics (San Carlos, Calif.). Cysteine end terminalaffibody was purchased from Affibody® (Sweden). All other reagents werepurchased from Sigma Aldrich.

Maleimide-poly(ethylene glycol)-block-poly(_(D,L)-lactic acid)(MAL-PEG-PLA), COOH-poly(ethylene glycol)-block-poly(_(D,L)-lactic acid)(COOH-PEG-PLA), and methoxypoly(ethyleneglycol)-block-poly(_(D,L)-lactic acid) (mPEG-PLA) were synthesized byring opening polymerization in anhydrous toluene using tin(II)2-ethylhexanoate as catalyst. General procedure for syntheses of thecopolymers is as follows: _(D,L)-Lactide (1.6 g, 11.1 mmol) andMAL-PEG₃₅₀₀-OH (0.085 mmol) or COOH-PEG₃₅₀₀-OH (0.085 mmol) in anhydroustoluene (10 mL) was heated to reflux temperature (ca. 120° C.), afterwhich the polymerization was initiated by adding tin(II)2-ethylhexanoate (20 mg). After stirring for 9 h with reflux, thereaction mixture was cooled to room temperature. To this solution wasadded cold water (10 mL) and then resulting suspension was stirredvigorously at room temperature for 30 min to hydrolyze unreacted lactidemonomers. The resulting mixture was transferred to separate funnelcontaining CHCL₃ (50 mL) and water (30 mL). After layer separation,organic layer was collected, dried using anhydrous MgSO₄, filtered, andconcentrated under reduced vacuum. Then, hexane was added to theconcentrated solution to precipitate polymer product. PureMAL-PEG₃₅₀₀-PLA or COOH-PEG3500-PLA was collected as a white solid.111PEG2000-PLA was also prepared by same procedure above. Bothcopolymers were characterized by ′H-NMR (400 MHz, Bruker Advance DPX400) and gel permeation chromatography (GPC) (Waters Co, Milford, Mass.,USA). Alternatively, the conjugation of PLGA or PLA and PEG was achievedin the presence of 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride) (EDC) and N-hydroxysuccinimide (NHS). Briefly, PLGAparticles were dissolved in acetonitrile. The carboxylic end of PLGA wasactivated by mixing with NHS and EDC at a molar ratio of COOH to EDC andNHS and stir overnight at room temperature. The excess EDC and NHS inthe solution were quenched by adding 2-mercaptoethanol. The NHSactivated PLGA was purified by precipitation in a solution containingethyl ether and methanol, and followed by centrifugation at 3000 g for10 minutes. To conjugate the amine end of NH2-PEG-MAL with theNHS-activated PLGA, both polymers mixed at a molar ratio of 1:1.3(PLGA-NHS:NH2-PEG-MAL) at room temperature overnight. The resultingPLGA-PEG-MAL copolymer was purified by precipitation in ethylether-methanol solution. The conjugation of the maleimide end ofMAL-PEG-PLGA and the free end thiol of affibody.

Nanoparticles were formed by precipitating the triblock copolymer inwater. Briefly, the triblock polymer was dissolved in acetonitrile, andthen mixed slowly with water. The nanoparticles formed instantly uponmixing. The residual acetonitrile in the suspension was evaporated bycontinuously stirring the suspension at room temperature for 4 hrs.Alternatively, polymeric nanoparticles were formed in a first step asabove and subsequently functionalized with affibody in aqueous solution.

Anti-Her2 Affibody Targeted Polymeric Nanoparticles

The synthesis of a multi-block polymer is initiated by conjugation offunctionalized biodegradable polyesters with chemical groups such as,but not limited to, maleimide or carboxylic acid for easy conjugation toone end of thiol, amine or similarly functionalized polyethers. Theconjugation of polymer to the affibody will be performed in organicsolvents such as but not limited to dimethyl sulfoxide, dichloromethane,acetonitrile, chloroform, dimethylformamide, tetrahydrofuran, andacetone or in aqueous buffer including phosphate buffers and Trisbuffers. The other free end of the polyether would be functionalizedwith chemical groups for conjugation to a library of targeting moleculessuch as affibodies, and its derivatives. The affibody may be conjugatedthrough functional group including but not limited to thiol, amine,carboxylates, hydroxyls, aldehydes, ketones and photoreactions. Theconjugation reaction between the targeting molecules and thepoly-ester-ether copolymer is achieved by adding the affibody moleculessolublized in an organic solvent or aqueous solution. Following each ofthe two conjugation reactions, unconjugated macromers are washed away byprecipitating the polymer of interest in solvents such as but notlimited to ethyl ether, hexane, methanol and ethanol. Alternatively, thenanopartciles conjugated to affibody in aqueous solution are washedusing distilled water and ultracentrifuge membranes. Biodegradable andbiocompatible polymer poly(lactide-co-glycolide) (PLGA)/PLA andpolyethylene glycol (PEG) can be used as a model for the block copolymerof poly(ester-ether). In a representative embodiment, the humanepidermal growth factor receptor 2 (HER-2/neu, also known as erbB-2) canbe used for breast or ovarian specific targeting using an anti-HER-2Affibody as the targeting molecule to cancer cells. Carboxylic acidmodified PLGA (PLGA-COOH) or PLA can be conjugated to the amine modifiedheterobifunctional PEG (NH2-PEG-Maleimide) and form a copolymer ofPLGA-PEG-COOH. By using a C-end terminal cysteine modified Anti-Her2affibody (HS-Affibody). A triblock copolymer of PLGA/PLA-PEG-Affibodycan be obtained by conjugating the maleimide end of PEG and free thiolfunctional group on the affibody. The multiblock polymer can also beuseful for imaging and diagnostic applications. In such embodiment, aphoto-sensitive or environmental-responsible compound will be linked tothe multiblock polymer.

The targeted nanoparticles are formed by precipitation of themulti-block polymer in an aqueous environment. The nanoparticleformulation system described here is compatible with high throughputbiological assays in order to test the nanoparticles generated from themulti-block polymer. Alternatively, polymeric nanoparticles can beformed by nanoprecipitation and subsequently functionalized with theaffibody in aqueous solution. It is possible to control the density ofaffibody on the surface and to optimize the formulation polymer/affibodyfor therapeutic application.

AKERC Peptide Targeted Lipid-Polymer Nanoparticles

The peptide is first chemically conjugated to the hydrophilic region ofa lipid molecule. This conjugate is then mixed with a certain ratio ofunconjugated lipid molecule in an aqueous solution containing one ormore water-miscible solvents. In a preferred embodiment, the amphiphiliclipid can be, but is not limited to, one or a plurality of thefollowing: phosphatidylcholine, lipid A, cholesterol, dolichol,shingosine, sphingomyelin, ceramide, cerebroside, sulfatide,glycosylceramide, phytosphingosine, phosphatidylethanolamine,phosphatidylglycerol, phosphatidylinositol, phosphatidylserine,cardiolipin, phophatidic acid, and lysophophatides. The water misciblesolvent can be, but is not limited to: acetone, ethanol, methanol, andisopropyl alcohol. Separately, a biodegradable polymeric material ismixed with the agent or agents to be encapsulated in a water miscible orpartially water miscible organic solvent. In a preferred embodiment, thebiodegradable polymer can be, but is not limited to one or a pluralityof the following: poly(D,L-lactic acid), poly(D,L-glycolic acid),poly(s-caprolactone), or their copolymers at various molar ratios. Thecarried agent can be, but is not limited to, one or a plurality of thefollowing: therapeutic drugs, imaging probes, or hydrophobic orlipophobic molecules for medical use. The water miscible organic solventcan be but is not limited to: acetone, ethanol, methanol, or isopropylalcohol. The partially water miscible organic solvent can be, but is notlimited to: acetonitrile, tetrahydrofuran, ethyl acetate, isopropylalcohol, isopropyl acetate, or dimethylformamide. The resulting polymersolution is then added to the aqueous solution of conjugated andunconjugated amphiphilic lipid to yield nanoparticles by the rapiddiffusion of the organic solvent into the water and evaporation of theorganic solvent.

In a preferred embodiment, the peptide AKERC, which binds to collagen IVin the extracellular basement membranes, is conjugated toDSPE-PEG-Maleimide (DSPE: 1,2distearoyl-sn-glycero-3-phosphoethanolamine sodium salt) using EDC/NHSchemistry with a conjugate concentration of 0.7 mg/mL. 0.21 mg of thisDSPE-PEG-KAERC bioconjugate is mixed with 0.07 mg lecithin in 2 mLaqueous solution containing 4% ethanol. 1 mg poly(D,L-lactic-co-glycolicacid) (PLGA, Mw=100 kD) is dissolved in 1 mL acetonitrile (ACN) solvent,to which 5% docetaxel of the mass of PLGA is added. This PLGA solutionis then mixed with the aqueous solution of lecithin/DSPE-PEG-KAERC.These mixtures are vortexed for 3 minutes, followed by stirring for 2hours. In order to remove all organic solvents, these mixtures are thenwashed three times using copious PBS buffer. This peptide will targetthe delivery and uptake of the nanoparticles to extracellular basementmembranes which are exposed under the leaky endothelial layer coveringatherosclerotic plaques.

Example 5 CREKA-Targeted Nanoparticle Lipid-PEG-CREKA Synthesis:

1 mg CREKA peptide is dissolved in 0.2 mL PBS buffer containing 10 mgdithiothreitol (DTT). The solution is incubated at room temperature for30 minutes before mixed with 1 mg DSPE-PEG-Maleimide (DSPE: 1,2distearoyl-sn-glycero-3-phosphoethanolamine sodium salt). These mixturesare incubated at 4° C. for 24 hr. In order to remove DTT agent and theextra CREKA, these mixtures are then dialyzed against PBS buffer for 48hr.

CREKA-Targeted Nanoparticle Synthesis:

0.03 mg of the DSPE-PEG-CREKA bioconjugate is mixed with 0.07 mglecithin in 2 mL aqueous solution containing 4% ethanol. 1 mgpoly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved in 1 mLacetonitrile (ACN). This PLGA solution is then mixed with the aqueoussolution of lecithin/DSPE-PEG-CREKA. These mixtures are vortexed for 3minutes, followed by stirring for 2 hours. In order to remove allorganic solvents, these mixtures are then washed three times usingcopious PBS buffer. For fluorescence imaging purposes, 10% of the PLGApolymer is labeled with a fluorescent probe such as Alexa Fluor 647.

CREKA-Targeted Nanoparticle with Therapeutic Agent:

For example, 0.03 mg DSPE-PEG bioconjugate is mixed with 0.07 mglecithin in 2 mL aqueous solution containing 4% ethanol. 1 mgpoly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved in 1 mLacetonitrile solvent, to which 5% docetaxel of the mass of PLGA isadded. The lecithin/DSPE-PEG solution is first heated up to 65° C. for 3minutes. Then the PLGA solution is added to the aqueous solution oflecithin/DSPE-PEG dropwise under gentle stirring. These mixtures arevortexed for 3 minutes, followed by stirring for 2 hours. In order toremove all organic solvents, these mixtures are then dialyzed foranother 3 hours against PBS buffer. These procedures would yieldlipid-polymer hybrid nanoparticles with a diameter of about 50-60 nm anda zeta potential of about −40 mV.

In another method, 0.036 mg DSPE-PEG-CREKA triblock compound is mixedwith 0.07 mg lecithin in 2 mL aqueous solution containing 4% ethanol. 1mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved in 1mL acetonitrile solvent, to which 5% docetaxel of the mass of PLGA isadded. For fluorescence imaging purpose, 10% of the PLGA polymer islabeled with a fluorescent probe such as Alexa Fluor 647. Thelecithin/DSPE-PEG solution is first heated up to 65° C. for 3 minutes.Then the PLGA solution is added to the aqueous solution oflecithin/DSPE-PEG dropwise under gentle stirring. These mixtures arevortexed for 3 minutes, followed by stirring for 2 hours. In order toremove all organic solvents, these mixtures are then dialyzed foranother 3 hours against PBS buffer. Alternatively, these mixtures can bewashed three times using copious PBS buffer to remove organic solventsand any free molecules. These procedures would yield CREKA-targetedlipid-polymer nanoparticles with specific a binding affinity toextracellular basement membrane.

In another method, 0.03 mg DSPE-PEG-Maleimide bioconjugate is mixed with0.07 mg lecithin in 2 mL aqueous solution containing 4% ethanol. 1 mgpoly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) is dissolved in 1 mLacetonitrile solvent, to which 5% docetaxel of the mass of PLGA isadded. For fluorescence imaging purpose, 10% of the PLGA polymer islabeled with a fluorescent probe such as Alexa Fluor 647. TheLecithin/DSPE-PEG solution is first heated up to 65° C. for 3 minutes.Then the PLGA solution is added to the aqueous solution oflecithin/DSPE-PEG dropwise under gentle stirring. These mixtures arevortexed for 3 minutes, followed by stirring for 2 hours. In order toremove all organic solvents, these mixtures are then dialyzed foranother 3 hours against PBS buffer. Alternatively, these mixtures can bewashed three times using copious PBS buffer to remove organic solventsand any free molecules. 0.03 mg CREKA peptide is dissolved in 0.2 mL PBSbuffer containing 1 mg dithiothreitol (DTT). The solution is incubatedat room temperature for 30 minutes before mixed with 1 mgPLGA-Lipid-PEG-Maleimide nanoparticle. These mixtures are incubated at4° C. for 24 hr. In order to remove DTT agent and the extra CREKA, thesemixtures washed three times using PBS buffer. These procedures wouldyield CREKA-targeted lipid-polymer nanoparticles with specific a bindingaffinity to extracellular basement membrane.

In another method, 0.036 mg DSPE-PEG-CREKA triblock compound can bemixed with 0.07 mg lecithin in 2 mL aqueous solution containing 4%ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) can bedissolved in 1 mL acetonitrile solvent, to which 5% of a combination ofdexamethasone and zotarolimus of the mass of PLGA can be added.

In another method, 0.036 mg DSPE-PEG-CREKA triblock compound can bemixed with 0.07 mg lecithin in 2 mL aqueous solution containing 4%ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) can bedissolved in 1 mL acetonitrile solvent, to which 5% of everolimus of themass of PLGA can be added.

In another method, 0.036 mg DSPE-PEG-CREKA triblock compound can bemixed with 0.07 mg lecithin in 2 mL aqueous solution containing 4%ethanol. 1 mg poly(D,L-lactic-co-glycolic acid) (PLGA, Mw=100 kD) can bedissolved in 1 mL acetonitrile solvent, to which 5% of paclitaxel of themass of PLGA can be added.

ARYLQKLN-Targeted Nanoparticle Synthesis:

For ease of conjugation, a cystin amino acid is added to the C-terminalor N-terminal of ARYLQKLN. The additional procedures/methods followthose of CREKA-targeted nanoparticles given immediately above.

CREKA-Targeted Nanoparticle Binding to Collagen-Coated Surface

To prepare a collagen IV-coated surface, 0.5 mL Collagen IV acetic acidsolution (10 μg/mL) is spread to completely cover the bottom of a glasswell. After 1 hr incubation at 37° C., the extra collagen is removed bywashing the surface using copious water. The quality of the coating ischecked by atomic force microscopy (AFM). The collagen coated surface isthen incubated with 1 mL CREKA-targeted nanoparticle aqueous solution(0.25 mg/mL) for 30 minutes at room temperature. The extra nanoparticlesare washed by copious PBS buffer. Fluorescence microscopy is used toimage the sample, thereby identifying the binding efficiency ofCREKA-targeted nanoparticle to collagen coated surface.

CREKA-Targeted Nanoparticle Binding to Rat Basement Membrane Ex Vivo:

A rat is sacrificed and its abdominal aorta is exposed in situ. Afterwashing the aorta with copious PBS buffer, a balloon catheter is placedin the aorta. The balloon is inflated with 0.2 mL air and draggedthrough the aorta five times to injure the endothelia layer. The properamount of CREKA-targeted nanoparticles are injected into the aorta andincubated for 30 minutes under constant pressure. The extrananoparticles are washed away using copious PBS buffer. 10 mm abdominalaorta is sectioned and kept in 4% formaldehyde solution for histologicalimaging use.

Other peptides such as d-CREKA and CEAKR (a scrambled sequence) are usedas negative control to investigate the binding specificity ofCREKA-targeted nanoparticles to basement membrane. d-CREKA and CEAKRreplace CREKA and repeated in the above experiments.

Example 6 Prophetic Example of Synthesis of PLGA-PEG-CREKA Macromoleculefor Preparation of CREKA-Targeted Nanoparticle

Example 7 Local Delivery of Nanoparticle

As described herein, the nanoparticles of the invention can be deliveredto a subject using a variety of methods, such as intravenous or localadministration using, e.g., a balloon stent. The following exampledemonstrates how to test the advantages of locally delivering thenanoparticles of the invention.

Pre-Interventional Procedures

Animal Monitoring and Examination

Upon arrival at the Testing Facility and until sacrifice, the animalswill be monitored and observed at least once a day. A physicalexamination of all animals entered in the study will be done by theFacility Veterinarian or a trained employee during acclimation, as perTesting Facility SOPs.

Fasting

Fasting will be conducted prior to induction of anesthesia. Food,including any dietary supplements, will be withheld the morning of theprocedure. Water will not be withheld.

Anesthesia

Animals will be tranquilized with acepromazine administeredsubcutaneously [SC]. Animal weight will be recorded. Anesthesiainduction will be achieved with propofol injected intravenously [IV].Upon induction of light anesthesia, the subject animal will be intubatedand supported with passive balloon ventilation. Isoflurane in oxygenwill be administered to maintain a surgical plane of anesthesia. Fluidtherapy will be achieved by saline injections before surgery.Intravenous saline injection may be performed to replace blood loss orto correct low systemic blood pressure.

Anticoagulant Therapy

During the procedure an initial bolus of heparin (˜70 IU/kg) will begiven following cannulation of the carotid artery. Additional heparinmay be given if needed.

Animal Preparation

The animal will be placed in dorsal recumbency, and the hair will beremoved from the access areas. A rectal temperature probe will beinserted, and the temperature will be monitored regularly. The accesssite will be prepared with topical application of chlorhexidine, 70%isopropyl alcohol and proviodine. The area will then be appropriatelydraped to maintain a sterile field.

Denudation Procedures

Vascular Access

After induction of anesthesia, the left or right carotid artery will beaccessed with an incision made in the throat region. An application ofbupivacain on the carotid access site will be performed to achieve localanesthesia and manage pain after surgery. An arterial sheath will beintroduced and advanced into the artery.

Vessel Angiography

Before the first angiogram, 1 ml of nitroglycerine (0.5 mg/ml) IV willbe given. For subsequent angiograms, more nitro can be given if slowflow or spams are observed, as per operator judgment and animalcondition. The iliac artery will be circumscribed (from the femoral tothe internal iliac branch) and Quantitative Angiography (QA) will beperformed to document the vessel size. Extra angiograms may be recordedat this point or later on in the procedure at the discretion of theoperator. In such case, the operator will select a suitable angiogramfor analysis.

Balloon Injury

The appropriate balloon will be advanced over the guidewire to traversethe distal portion of the pre-selected injury site. The inflated balloonwill then be retracted from the femoral artery back into the aorta toenable denudation of the target vasculature. The balloon will bedeflated and re-advanced to traverse the target injury site. At theoperator's discretion, the inflation pressure will be adjusted for eachsubsequent denudation pass, based on the amount of force required topull the balloon. If there is little resistance to the pulling, theballoon inflation pressure will be increased by an increment of one Atm.If there is too much resistance, the balloon inflation pressure will bedecreased by an increment of one ATM. The third denudation will not beperformed if resistance is still present. The balloon will be deflatedwhile it is in the terminal descending aorta and the denudationprocedure repeated one more time (total 3 times denudation).Post-denudation angiogram will be performed and TIMI flow will beassessed. Animals with post-TIMI flow of zero or 1 will receiveintra-arterial infusion of nitroglycerine (at the discretion of theinterventionalist) to restore the flow to 2 or 3.

Test Article Delivery

The delivery catheter will be introduced into the artery over the guidewire. The Genie balloon catheter will be continuously inflated at a lowpressure of 2 atm that allows for distal and proximal occlusion of thevessel while simultaneously forming a central drug depot. A continuouspressure of 2 atm is maintained throughout application of the contrastagent. The total volume injected will be recorded. Following treatmentof the vessel a final angiography will be taken and recorded, TIMI flowand QCA will also be performed and documented.

Monitoring Procedures

Parameters including isoflurane level, blood oxygen saturation, pulserate, and temperature will be regularly monitored and manually recordedand noted in the raw data for each animal.

Closure

Following successful delivery and completion of angiography, allcatheters and the sheath will be removed from the animals and thecarotid artery will be ligated.

Necropsy

Upon completion of follow-up angiography the animals will be kept deeplyanesthetised before euthanasia with a rapid bolus of pentobarbital.

Stented arteries along with proximal and distal non-stented segmentswill then be dissected out rinsed and immersion-fixed inneutral-buffered formalin and processed for histology.

Histopathology

The treated iliacs will be cut in 4 sections that will be embedded inparaffin 4 separate blocks. For each block 2 adjacent sections will beprepared and then 2 other adjacent section ˜100μ deeper in the block.One section from each adjacent pair will be stained with hematoxylin andeosin (H&E). The remaining section of each adjacent pair will be leftunstained and sent for fluorescence analyses. The H&E stained sectionwill be analyzed by the Study Pathologist for the extent of vesselinjury, the presence of endothelium and other relevant observations. Theanalysis will be reported as a narrative text including representativeimages, with scoring of some parameters if deemed appropriate.

Example 8 HER-2 Targeted Drug Encapsulated NanoParticles (FIGS. 21-24)

To develop HER-2 targeted drug encapsulated NPs, the anti-HER-2 Affibodywas conjugated to the thiol-reactive maleimide end group of thePLA-PEG-Maleimide (PLA-PEG-Mal) copolymer through a stable thioetherbond and the targeting specificity and efficacy was evaluated usingfluorescent microscopy. Subsequently, we encapsulated paclitaxel intothe targeted polymeric NPs and examined whether this system couldincrease the drug cytotoxicity in HER-2 positive cell lines: SK-BR-3 andSK-OV-3.

Materials and Methods

Conjugation and Characterization of nanoparticle-Affibody bioconjugates:PLA-PEG-Mal polymeric NPs were incubated under stirring conditions withthe Anti-HER-2 Affibody molecules (15 kDa) at a molar ratio ofAffibody:PLA-PEG-Mal of 5% to form a stable bioconjugate. TheNP-Affibody bioconjugates were purified to remove free Affibodymolecules using Amicon Ultra centrifuge device (100 kDa molecular weightsize exclusion). Subsequently, the thioether bond formation between thePLA-PEG-Mal NPs and the Affibody molecules was characterized usingproton nuclear magnetic resonance (¹H-NMR, 600 MHz, Bruker Advance).Additionally, the chemical attachment of the fluorescent Affibody wasconfirmed using Ultra Violet Imaging system (Kodak ElectrophoresisDocumentation and Analysis System 120). The Affibody molecule wasfluorescently labeled with a red fluorescent probe, Alexa Fluor 532(Invitrogen), purified and subsequently conjugated to PLA-PEG-Malpolymeric NPs at different molar ratios of Affibody:PLA-PEG-Mal rangingfrom 0 to 20% (molar ratio). Then the purified NP-Affibody bioconjugatessuspensions were imaged using a UV Kodak camera assisted with a redfilter to show the visible effect of the fluorescent Affibody conjugatedon non-fluorescent polymeric NPs.

Uptake assays of targeted and untargeted nanoparticles: The humanovarian adenocarcinoma (SK-OV-3; ATCC), human breast adenocarcinoma(SK-BR-3; ATCC), and human pancreatic adenocarcinoma (Capan-1, ATCC)were the HER-2 positive cell lines used for cytotoxicity and uptakeefficacy studies of the NP-Affibody bioconjugates. HER-2 positive celllines were grown in chamber slides (Cab-TekII, 8 wells; Nunc) withintheir growth medium (Modified McCoy's 5a (ATCC) supplemented with 100units/ml aqueous penicillin G, 100 ug/ml streptomycin, and 10% FBS) to70% confluence in 24 h (i.e., 50,000 cells/cm²) in 5% CO₂ incubator. Onthe day of the experiment, cells were washed with pre-warmed PBS andincubated with pre-warmed phenol-red-reduced OptiMEM media for 30minutes, before adding 50 μg of NPs or NP-Affibody bioconjugates loadedwith same amount of fluorescent NBD dye. NP formulations were incubatedwith cells for 2 hours at 37° C., washed with PBS three times, fixedwith 4% paraformaldehyde, blocked for 30 minutes at room temperaturewith 1% BSA/PBS, permeabilized with 0.01% Triton-X for 3 minutes,counterstained with Alexa-Fluor Phalloidin-Rhodamine (cytoskeletonstaining), 4′,6-diamidino-2-phenylindole (DAPI, nucleus staining),mounted in fluorescence protecting imaging solution, and visualizedusing fluorescent microscopy (DeltaVision system; Olympus IX71). Digitalimages of green, red and blue fluorescence were acquired along thez-axis at 0.2 μm intervals using 60× oil immersion objective and DAPI,FITC and Rhodamine filters respectively. Images were overlaid,deconvoluted and 3D reconstruction was performed using Softwork softwarefor acquisition and analysis.

In vitro cellular toxicity assay of paclitaxel encapsulated intotargeted and untargeted NPs: SK-BR-3 and SK-OV-3 were grown in 96-wellplates at concentrations leading to 70% confluence in 24 h (i.e., 50,000cells/cm²). Defined amounts of paclitaxel were encapsulated intotargeted and non-targeted nanoparticles and incubated with cell lines (5ug Paclitaxel/well) in OptiMEM for two hours. Next, cells were washedand fresh media was supplemented. The cells were then allowed to growfor 72 hours and cell viability was assessed colorimetrically with MTSreagents (Invitrogen).

Results

Development of targeted, controlled release drug delivering NP-Affibodybioconjugates. We first synthesized a copolymer comprised of ahydrophobic block, poly(_(D,L)lactic acid), and a hydrophilic block,poly(ethylene glycol) with a maleimide terminal group (PLA-PEG-Mal).Then the copolymers form negatively charged NPs with a core-shellstructure in aqueous environment via the nanoprecipitation method. Thehydrophobic core of the NPs is capable of carrying pharmaceuticals,especially poorly soluble drugs. The hydrophilic shell not only providesa “stealth” layer, together with the surface charge property (Zetapotential)=−10 mV±5 mV), to improve the stability and the circulationhalf-time of these drug delivering NPs, but also functional maleimidegroups for Affibody conjugation. Lack of protein adsorption in solutionsincluding 10%, 20% and 100% serum demonstrated the stability of NP size(<100 nm). We also evaluated the freeze-drying process for storing thenanoparticles in a dry state, as described previously. We were able toreconstitute nanoparticles with a similar original size afterlyophilization, confirming the stability of this type of carrier to thisprocess.

The anti-HER-2 Affibody molecule was previously selected against theextracellular domain of the HER-2 protein and further modified byaffinity maturation and dimerization. The anti-HER-2 Affibody iscommercially available and has been shown to have high bindingspecificity and affinity in vitro and in vivo as a targeted imagingagent. Particle size and surface charge (Zeta potential) of PLA-PEG-MalNPs both with and without Affibody were characterized using laser lightscattering, ZetaPALS system and electron microscopy (FIGS. 21A and 21B).The addition of Affibody molecules on the surface of the NPs did notsignificantly affect the size, size distribution and surface charge ofthe NPs (NP=70±5 nm, NP-Affibody 85±5 nm). The chemical conjugation ofthe Affibody molecules on the surface of the PLA-PEG-Mal NPs wasconfirmed using UV imaging (FIGS. 21A and 21B) and proton nuclearmagnetic resonance spectroscopy in d-DMSO (¹H-NMR) (FIG. 22C). Tovisualize the presence of Affibody molecules on the NPs, we labeledAffibody molecules with fluorescence probe, Alexa Fluor 532, andsubsequently conjugated them to the PLA-PEG-Mal NPs with different molarratios of Affibody:PLA-PEG-Mal (0, 1, 2, 5, 20%). The NP-Affibodybioconjugates were then exposed under a UV lamp to observe theirfluorescence signals. No fluorescence signal was observed from the NPswithout fluorescently labeled Affibody, however, the fluorescenceintensity from those NPs with fluorescent Affibody continuously enhanceswith the increase of Affibody:PLA-PEG-Mal molar ratio. The ¹H-NMRspectrum of the purified PLA-PEG-Affibody in d-DMSO showed thecharacteristic peaks of PLA-PEG at chemical shift of δ˜1.4 ppm (—CH3 ofthe PLA backbone), δ˜3.6 ppm of (—CH₂ of the PEG backbone) and δ˜5.2 ppm(—CH of the PLA backbone). Additionally, we observed the characteristicpeaks of the Affibody molecule in the chemical shift region of δ=7-8 ppmthat represents the amide bonds (NH—CO) within the Affibody polypeptidemolecule. The NMR results suggest successful conjugation of the Affibodyon the surface of PLA-PEG-Mal NPs.

Efficient and specific receptor mediated internalization of NP-Affibodybioconjugates. We next demonstrated the efficient binding andinternalization of targeted NP-Affibody bioconjugates to HER-2 positivecancer cells using three cell lines: Capan-1, SK-BR-3, and SK-OV-3 (seeFIG. 22). After incubating NBD dye encapsulated NP-Affibodybioconjugates with the cells for 2 hr at 37° C. and removing the excessbioconjugates, we observed a large amount of green dots in a punctuatepattern inside the targeted cells, suggesting an efficient targeting andinternalization mechanism of the ˜80 nm NP-Affibody bioconjugates to theHER-2 positive cells. In contrast, untargeted PLA-PEG NPs were slightlytaken up by the cell lines after the same duration of incubation (FIG.22). To minimize cell passage effect on the observed results, thisexperiment was repeated four times with different cell passages and allof them gave the same observations. We also verified the cellularlocalization of the NP-Affibody bioconjugates using a z-axis scanningfluorescent microscopy and 3D image reconstitution. The rotated crosssection of the 3D reconstitution images of a SK-BR-3 cell demonstratedthe internalization of targeted NP-Affibody bioconjugates to the cell(FIG. 23). Orlova et al. have shown the binding ability of Anti-HER-2Affibody within 1 hr using immunofluorescence method. Our results areconsistent with their findings and suggest a receptor mediatedendocytosis mechanism. Internalization through an endocytosis mechanismhas been previously described for anti-HER-2 monoclonal antibodies andis consistent with the kinetics of our NPs entering the cells.Similarly, targeted drug delivery using RGD peptide sequences tointegrins has also shown efficient binding and internalization inmultiple types of cancers. In contrast, the anti-HER-2 approach offersbetter cancer diseases specificity with high affinity to HER-2 cellmembrane receptors expressed in multiple types of cancers.

In vitro cellular cytotoxicity assays using breast cancer and ovariancancer cells (MTS assays). We prepared targeted and untargeted NPs (withand without paclitaxel) to evaluate their differential cytotoxicityusing in vitro cell viability assay (MTS assays) with breast cancer andovarian cancer cells (SK-BR-3; SK-OV-3), which over-express the HER-2cell membrane receptors. In this study, we incubated various NPformulations with SK-BR-3 and SK-OV-3 cancer cells for 2 hours inoptimem, washed cells with PBS to remove excess of NPs, and supplementedwith fresh cell growth medium. We further incubated the cells for 3 daysbefore using MTS assay to quantify cell viability which was normalizedto that of the cells in the absence of NPs. The results showed that drugencapsulated targeted NPs had the highest cytotoxicity to both SK-BR-3and SK-OV-3 cell lines; cell viability was 70±5% and 59±5%, respectively(FIGS. 24A and 24B). The ANOVA test indicated that the cell viability oftargeted NPs differed significantly from that of untargeted NPs(p<0.05). In contrast, NPs without encapsulated drugs are not toxic toboth cell lines. These results are consistent with our previous studiesusing targeted NP-aptamer bioconjugates to deliver drugs to prostatecancer cells. Therefore, this NP-Affibody bioconjugate system holdsgreat potential to be used as a biocompatible and biodegradable targeteddrug delivery platform for multiple types of cancers therapy. For aspecific application, it would be feasible to tune some parameters ofthe bioconjugates such as NP size, surface charge and Affibody packingdensity to optimize the drug delivery pharmacokinetics and its targetingefficiency.

FIG. 10 demonstrates a schematic illustration of a CREKA-targetedPLGA-Lipid-PEG nanoparticle.

FIGS. 11A and 11B demonstrate that (A) CREKA-targeted PLGA-Lipid-PEGnanoparticles effectively bind to collagen IV coated surface. Forfluorescence imaging purpose, fluorescent probe Alexa 647 was chemicallyconjugated to PLGA polymer; (B) a bare (nontargeted) PLGA-Lipid-PEGnanoparticles rarely bind to collagen IV coated surface.

FIGS. 12A and 12B demonstrate (A) H&E staining of normal rat aorta; (B)H&E staining of balloon injured aorta; endothelium layer was removed.

FIGS. 13A and 13B demonstrates CREKA-targeted PLGA-Lipid-PEGnanoparticles effectively bind to balloon-injured rat aorta. Thenanoparticles were incubated with the aorta for 10 minutes. The extrananoparticles were washed away with copious PBS buffer. Then the aortawas harvested, fixed and prepared for imaging. (A) Fluorescence image ofthe aorta; (B) Overlay of fluorescence image and phase image of the sameaorta.

FIGS. 14A and 14B demonstrate that D-CREKA-targeted PLGA-Lipid-PEGnanoparticles (D-form of amino acids) do not bind to balloon-injured rataorta. The nanoparticles were incubated with the aorta for 10 minutes.The extra nanoparticles were washed away with copious PBS buffer. Thenthe aorta was harvested, fixed and prepared for imaging. (A)Fluorescence image of the aorta; (B) Overlay of fluorescence image andphase image of the same aorta.

FIGS. 15A and 15B demonstrate that scrambled peptide CEAKR-targetedPLGA-Lipid-PEG nanoparticles do not bind to balloon-injured rat aorta.The nanoparticles were incubated with the aorta for 10 minutes. Theextra nanoparticles were washed away with copious PBS buffer. Then theaorta was harvested, fixed and prepared for imaging. (A) Fluorescenceimage of the aorta; (B) Overlay of fluorescence image and phase image ofthe same aorta.

FIGS. 16A and 16B demonstrate that CREKA-targeted PLGA-Lipid-PEGnanoparticles do not bind to a normal rat aorta. The nanoparticles wereincubated with the aorta for 10 minutes. The extra nanoparticles werewashed away with copious PBS buffer. Then the aorta was harvested, fixedand prepared for imaging. (A) Fluorescence image of the aorta; (B)Overlay of fluorescence image and phase image of the same aorta.

FIG. 17 is a schematic illustration of CREKA-targeted PLGA-Lipid-PEGnanoparticle

FIG. 18 demonstrates fluorescence images of ARYLQKLN-targetedPLGA-Lipid-PEG nanoparticles incubating with basement membrane proteinsfor 10 minutes: (A) PBS; (B) Collagen I; (C) Collagen II; (D) CollagenIV; (E) Fibronectin; and (F) vitronectin.

FIG. 19 demonstrates that ARYLQKLN-targeted PLGA-Lipid-PEG nanoparticlesbind to a balloon-injured rat aorta. The nanoparticles were incubatedwith the aorta for 10 minutes. The extra nanoparticles were washed awaywith copious PBS buffer. Then the aorta was harvested, fixed andprepared for imaging. (A) Fluorescence image of the aorta; (B) Overlayof fluorescence image and phase image of the same aorta.

FIG. 20 demonstrates that ARYLQKLN-targeted PLGA-Lipid-PEG nanoparticlesdo not bind to normal rat aorta. The nanoparticles were incubated withthe aorta for 10 minutes. The extra nanoparticles were washed away withcopious PBS buffer. Then the aorta was harvested, fixed and prepared forimaging. (A) Fluorescence image of the aorta; (B) Overlay offluorescence image and phase image of the same aorta.

For FIGS. 24A and 24B: Cell viability assay (MTS assay) to evaluate thedifferential toxicity of targeted (Np-Affb) and untargeted nanoparticles(Np) with and without encapsulated paclitaxel (Ptxl). In this assay, thenanoparticle formulations were incubated for 2 hours, cells weresubsequently washed and incubated in cell growth media to allow theeffect of the drug on the cell cycles before quantifying thenanoparticle formulations toxicities against two cancer cell linesexpressing HER-2 (SK-BR-3 and SK-OV-3).ANNOVA test “*” p<0.01; “**”p<0.05.

1. A controlled-release system, comprising a plurality oftarget-specific stealth nanoparticles; wherein said nanoparticlescontain targeting moieties attached thereto, wherein the targetingmoiety is a poly(amino acid) that targets the basement membrane of ablood vessel.
 2. The controlled-release system of claim 1, wherein thenanoparticle has an amount of targeting moiety effective for thetreatment of vulnerable plaque in a subject in need thereof.
 3. Thecontrolled-release system of claim 1, wherein the nanoparticle has anamount of targeting moiety effective for the treatment of restenosis. 4.The controlled-release system of claim 1, wherein the nanoparticle hasan amount of targeting moiety effective for the treatment of cancer in asubject in need thereof. 5-6. (canceled)
 7. The controlled-releasesystem of claim 1, wherein the poly(amino acid) comprises natural aminoacids, unnatural amino acids, modified amino acids, or protected aminoacids.
 8. The controlled-release system of claim 1, wherein thepoly(amino acid) is selected from the group consisting of a protein,peptidomimetic, affibody or peptide.
 9. The controlled-release system ofclaim 1, wherein the poly(amino acid) binds to the basement membrane ofa blood vessel.
 10. The controlled-release system of claim 1, whereinthe poly(amino acid) binds to collagen.
 11. The controlled-releasesystem of claim 1, wherein the poly(amino acid) binds to collagen IV.12. (canceled)
 13. The controlled-release system of claim 8, wherein thepeptide comprises a sequence selected from the group consisting ofAKERC, CREKA, ARYLQKLN and AXYLZZLN, wherein X and Z are variable aminoacids.
 14. The controlled-release system of claim 1, wherein thenanoparticle comprises a polymeric matrix.
 15. The controlled-releasesystem of claim 14, wherein the polymeric matrix comprises two or morepolymers.
 16. The controlled-release system of claim 13, wherein thepolymeric matrix comprises polyethylenes, polycarbonates,polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, dextran or polyamines, orcombinations thereof. 17-19. (canceled)
 20. The controlled-releasesystem of claim 14, wherein at least one polymer is a polyester.
 21. Thecontrolled-release system of claim 20, wherein the polyester is selectedfrom the group consisting of PLGA, PLA, PGA, and polycaprolactones. 22.The controlled-release system of claim 20, wherein the polyester is PLGAor PLA.
 23. The controlled-release system of claim 14, wherein thepolymeric matrix comprises a copolymer of two or more polymers.
 24. Thecontrolled-release system of claim 23, wherein the copolymer is acopolymer of a polyalkylene glycol and a polyester.
 25. Thecontrolled-release system of claim 24, wherein the copolymer is acopolymer of PLGA and PEG.
 26. The controlled-release system of claim24, wherein the polymeric matrix comprises PLGA and a copolymer of PLGAand PEG.
 27. (canceled)
 28. The controlled-release system of claim 14,wherein the polymeric matrix comprises lipid-terminated PEG and PLGA.29-34. (canceled)
 35. The controlled-release system of claim 14, whereinthe nanoparticle has a ratio of ligand-bound polymer tonon-functionalized polymer effective for the treatment of cancer. 36.The controlled-release system of claim 14, wherein the nanoparticle hasa ratio of ligand-bound polymer to non-functionalized polymer effectivefor the treatment of a vulnerable plaque.
 37. The controlled-releasesystem of claim 14, wherein the polymers of the polymer matrix have amolecular weight effective for the treatment of cancer.
 38. Thecontrolled-release system of claim 14, wherein the polymers of thepolymer matrix have a molecular weight effective for the treatment ofvulnerable plaque. 39-42. (canceled)
 43. The controlled-release systemof claim 1, wherein the nanoparticle further comprises a therapeuticagent. 44-45. (canceled)
 46. The controlled-release system of claim 43,wherein the therapeutic agent is selected from the group consisting ofmitoxantrone and docetaxel.
 47. The controlled-release system of claim43, wherein the therapeutic agent is selected from the group consistingof VEGF, fibroblast growth factors, monocyte chemoatractant protein 1(MCP-1), transforming growth factor alpha (TGF-alpha), transforminggrowth factor beta (TGF-beta), DEL-1, insulin like growth factors (IGF),placental growth factor (PLGF), hepatocyte growth factor (HGF),prostaglandin E1 (PG-E1), prostaglandin E2 (PG-E2), tumor necrosisfactor alpha (THF-alpha), granulocyte stimulating growth factor (G-CSF),granulocyte macrophage colony-stimulating growth factor (GM-CSF),angiogenin, follistatin, and proliferin, PR39, PR11, nicotine,hydroxy-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors,statins, niacin, bile acid resins, fibrates, antioxidants, extracellularmatrix synthesis promoters, inhibitors of plaque inflammation andextracellular degradation, and estradiol.
 48. The controlled-releasesystem of claim 43, wherein the therapeutic agent is selected from thegroup consisting of everolimus, paclitaxel, zotarolimus, pioglitazone,BO-653, rosiglitazone, sirolimus, dexamethasone, rapamycin, tacrolimus,biophosphonates, estrogen, angiopeptin, statin, PDGF inhibitors, ROCKinhibitors, MMP inhibitors, 2-CdA, zotarolimus and dexamethasone.
 49. Amethod of treating breast cancer in a subject in need thereof,comprising administering to the subject an effective amount of thecontrolled-release system of claim
 1. 50-54. (canceled)
 55. A method oftreating vulnerable plaque in a subject in need thereof, comprisingadministering to the subject an effective amount of thecontrolled-release system of claim
 1. 56. A method of treatingrestenosis in a subject in need thereof, comprising administering to thesubject an effective amount of the controlled-release system of claim 1.57. The method of claim 55, wherein the controlled-release system islocally administered to a designated region of the blood vessel wherethe vulnerable plaque occurs.
 58. The method of claim 55, wherein thecontrolled-release system is administered via a medical device.
 59. Themethod of claim 58, wherein the medical device is a drug eluding stent,needle catheter, or stent graft. 60-62. (canceled)
 63. A method ofpreparing a stealth nanoparticle, wherein the nanoparticle has a ratioof ligand-bound polymer to non-functionalized polymer effective for thetreatment of a disease, comprising: providing a therapeutic agent;providing a first polymer; providing a poly(amino acid) ligand; reactingthe first polymer with the poly(amino acid) ligand to prepare aligand-bound polymer; and mixing the ligand-bound polymer with a second,non-functionalized polymer, and the therapeutic agent; such that thestealth nanoparticle is formed. 64-71. (canceled)
 72. A stealthnanoparticle, comprising a copolymer of PLGA and PEG; and a therapeuticagent; wherein said nanoparticle contains targeting moieties attachedthereto, wherein the targeting moiety comprises AKERC or CREKA.
 73. Astealth nanoparticle, comprising a polymeric matrix comprising a complexof a phospholipid bound-PEG and PLGA; and a therapeutic agent; whereinsaid nanoparticle contains targeting moieties attached thereto, whereinthe targeting moiety is a poly(amino acid). 74-78. (canceled)
 79. Acontrolled-release system, comprising a plurality of target-specificstealth nanoparticles; wherein said nanoparticles contain targetingmoieties attached thereto, wherein the targeting moiety is a poly(aminoacid).
 80. (canceled)
 81. The compounds:

wherein n is 20 to 1720; and

wherein R₇ is an alkyl groups, R₈ is an ester or amide linkage, X=0 to 1mole fraction, Y=0 to 0.5 mole fraction, X+Y=20 to 1720, and Z=25 to455.